Composition comprising an antisense sequence implicated in the regulation of angiogenesis

ABSTRACT

Therapeutic compositions used in the field of angiogenesis include nucleotide sequences of genes, the involvement of the genes in the angiogenesis mechanism having been demonstrated by the Applicant, and including the complementary sequences thereof, the antisense sequences of same, polypeptide sequences coded by the coding parts of the aforementioned genes and antibodies that are directed against the polypeptide sequences and also relate to genetically-modified cells that underexpress or overexpress the above-mentioned genes and to therapeutic compositions containing the cells, which are used to treat angiogenic disorders, and, moreover, relate to methods of diagnosing and/or prognosticating antigenic disorders and to novel methods of screening active compounds in the treatment of the disorders.

RELATED APPLICATIONS

This is a divisional of U.S. Ser. No. 10/934,998, filed Sep. 3, 2004,which is a continuation of International Application No. PCT/FR03/00695,with an international filing date of Mar. 4, 2003 (WO 03/074073,published Sep. 12, 2003), which is based on French Patent ApplicationNos. 02/02717, filed Mar. 4, 2002, and 02/04546, filed Apr. 11, 2002.

TECHNICAL FIELD

This disclosure relates to the field of pharmaceutical compositions thatare useful for the treatment of pathologies resulting from aderegulation of the angiogenesis mechanism.

The disclosure relates to compositions comprising on the one handsequences of new genes whose function had not been identified to dateand whose implication in the mechanism of angiogenesis was demonstratedfor the first time by the applicant and on the other hand gene sequencesat least one of the functions of which was previously identified butwhose implication as constitutive genes of the endothelial cells in themechanisms of angiogenesis was demonstrated for the first time in thestudies performed by the applicant in the framework of this disclosure.These genes are identified by their nucleotide sequences in the attachedsequence listing. This disclosure also relates to the polypeptidesequences of the factors coded by the genes which find their applicationin the clinical study of the angiogenesis process, the prognosis,diagnosis and treatment of pathologies linked to this process as well asin the implementation of pharmacological, pharmacogenomic andpharmacosignalitic trials.

BACKGROUND

Angiogenesis is a fundamental process by means of which new bloodvessels are formed. This process is essential in many normalphysiological phenomena such as reproduction, development andcicatrization. In these normal biological phenomena, angiogenesis isunder strict control, i.e., it is triggered during a short period ofseveral days then completely inhibited. However, many pathologies arelinked to an invasive and uncontrolled angiogenesis. Arthritis, forexample, is a pathology caused by damage to cartilage caused by invasiveneovessels. In diabetic retinopathy, invasion of the retina byneovessels leads to the patients' blindness; neovascularization of theocular apparatus represents the major cause of blindness and thisneovascularization dominates around twenty eye diseases. Lastly, thegrowth and metastasis of tumors are linked directly toneovascularization and are dependent on angiogenesis, and the tumoritself stimulates the growth of the neovessels. Moreover, theseneovessels present escape pathways, allowing metastatic tumor cells toreach the blood circulation and cause metastases in remote sites such asthe liver, lungs and bones.

In other pathologies such as cardiovascular diseases, peripheralarterial diseases, and vascular and cerebral lesions, angiogenesis canpresent an important therapeutic base. The promotion of angiogenesis inthe damaged sites can lead to the formation of blood neovessels lateraland alternative to the damaged vessels, thereby providing blood flowand, by consequence, oxygen and other nutritive factors required for thesurvival of the tissues in question.

The formation of neovessels by endothelial cells involves the migration,growth and differentiation of endothelial cells. The regulation of thesebiological phenomena are directly linked to gene expression. In the areaof angiogenesis, a constantly growing number of studies show that theregulation of angiogenesis is implemented via an equilibrium between thefactors acting directly on the endothelial cell. These factors can beangiogenic stimulants, on the one hand, such as, among others, VEGF,FGFs, IL-8, HGF/SF and PDGF. They can also be angiogenic inhibitors suchas, among others, IL-10, IL-12, gro-α and -β, platelet factor 4,angiostatin; the inhibitor derived from human chondrocyte,thrombospondin and the leukemia inhibitory factor. (Jensen, Surg.Neural., 1998, 49, 189-195; Tamatani et al., Carcinogenesis, 1999, 20,957-962; Tanaka et al., Cancer Res., 1998, 58, 3362-3369; Ghe et al.,Cancer Res., 1997, 57, 3733-3740; Kawahara et al., Hepatology, 1998, 28,1512-1517; Chandhuni et al., Cancer Res., 1997, 57, 1814-1819;Jendraschak and Sage, Semin. Cancer Biol., 1996, 7, 139-146; Majewski etal., J. Invest. Dermatol., 1996, 106, 1114-1119).

One of the mechanisms by which cells respond to external stimulus is therecruitment of chains constituted by a set of proteins which provide forthe relay of an external signal to the interior of the cells. Byproviding for the transduction of the extracellular signal, this chainchanges the intracellular environment thereby controlling genetranscription (reviews: Avruch, 1998, Mol. Cell. Biochem., 182, 31-48;Karin, 1998, Ann. NY Acad. Sci., 851, 139-146). A large number of theseprotein chains, and by consequence the signal pathways which are highlyconserved via evolution, are collectively designated the pathways of the“mitogenic agent activated protein kinases” (MAPK) (Gupta et al., 1996,EMBO J., 15, 2760-2770; Madhani and Fink, 1998, Trends Genet. 14,151-155). The classic MAPK pathway is triggered by the binding of thegrowth factors to their receptor on the cell surface leading to theactivation of the protein Ras, which is a GTPase. This pathway resultsin the activation of the protein kinases regulated by extracellularsignals (ERKs), leading to gene transcription and cell proliferation. Aparallel MAPK pathway is stimulated by stress factors such as osmoticshock, cytotoxic products, UV radiation or inflammatory cytokines. Thispathway results in the activation of the stress-activated proteinkinases known by the designation of kinases acting on the N-terminal ofc-Jun (SAPK/JNKs) (Karin, 1998, Ann. NY Acad. Sci. 851, 139-146). Asecond stress-activated pathway leads to the activation of MAPK p38. Theeffect of stress activation extends to the proliferation,differentiation and even the gene transcription leading to thetermination of this cellular cycle and/or apoptosis, depending on thecell type and the stimulus (Karin, 1998, Ann. NY Acad. Sci. 851,139-146).

Many studies have reported a role for MAPK 1 and 2 as well as MAPK p38in the transduction pathway of the signal induced by the angiogenic oranti-angiogenic factors during angiogenesis, but no role has beenreported for MAPK4 in this process (Tanaka et al., 1999, Jpn. J. CancerRes., 90: 647-654; Erdreich-Epstein et al., 2000, Cancer Res., 60:712-721; Gupta et al., 1999, Exp. Cell Res., 247: 495-504; Bais et al.,1998, Nature, 391: 24-25; Rousseau et al., Oncogene, 1997, 15:2169-2177; Shore et al., 1997, Placenta, 18: 657-665).

MAPK 4 is one of the members of the MAPK family. This kinasephosphorylates directly and thereby activates the kinases actingdirectly on the N-terminal c-Jun (JNK) in response to stress and/orinflammatory cytokines. MAPK 4 is expressed in different tissues,however there is seen an abundance of expression of this kinase in theskeletal muscles and the brain. Mice deficient in the gene of MAPK 4develop abnormal hepatogenesis and die in the embryogenic state on thefourth day. However, cell lines deficient in MAP 4 have been obtained.These lines are characterized by the absence of gene transcriptiondependent of JNK and the transcription factor AP-1. Moreover, Tlymphocytes deficient in MAPK 4 exhibit a decoupling of the productionof IL-2 subsequent to the activation of the T cell receptors, suggestinga key role for MAPK4/JNK in the inflammatory process. The mutation ofMAPK4 in certain carcinomas indicates that it can play a tumorsuppressor role. Although the control of the expression and activity ofMAPK is currently the object of intense analyses and studies, thesestudies involve an approach for developing an anti-inflammatory andanticancer therapy. However, the role of MAPK4 in angiogenesis has notbeen demonstrated.

Pedram et al. (Endocrinology, 2001, 142: 1578-86) showed that thenatriuretic peptide suppresses or inhibits the angiogenesis induced byVEGF; they also showed that the activation of the kinases actingdirectly on the N-terminal of c-Jun is an important state in theinduction of angiogenesis by VEGF. In opposition, Jimenez et al.,Oncogene, 2001, 20: 3443-3448) reported that the activation of thekinases acting on the N-terminal of c-Jun is necessary for theinhibition of neovascularization by thrombospondin 1.

Neither of these studies reported a specific role of MAP4K4 in theregulation of angiogenesis.

The G proteins (proteins binding guanine) play a major role in thetransmembrane signaling pathways by transmission of extracellularsignals via the transmembrane receptors to their appropriateintracellular effectors (Gilman, 1987, Ann. Rev. Biochem., 56, 615-649;Simon et al., 1991, Science, 252, 802-808). After binding of the ligand,the receptor catalyzes the exchange of the GDP for a GTP in the alphasubunit of the heterotrimer G protein which induces its activation andthe dissociation of the alpha-GTP subunit from the beta and gammasubunits (Gilman, 1987, Ann. Rev. Biochem., 56, 615-649). TheG-protein-dependent signaling pathways are designated for amplifying andintegrating a multiplicity of both stimulatory and inhibitory responses,and their importance in cell function is such that they are tightlyregulated. PHLP (phosducin-like protein) is one of these regulatoryelements; it belongs to the family of phosducins and its isoforms,proteins that bind the G protein beta/gamma subunits, thereby blockingtheir function (Lee et al., 1987, Biochemistry 26, 3983-3990; Miles etal., 1993, Proc. Natl. Acad. Sci. USA, 90, 10831-10835; Craft et al.,1998, Biochemistry 37, 15758-15772). It has been proposed thatphosducin, strongly expressed in the photoreceptor cells of the retina(Lee et al., 1987, Biochemistry 26, 3983-3990, Wilkins et al., 1996, J.Biol. Chem., 271, 19232-19237) intervenes in the adaptation to light(Willardson et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 1475-1479). Incontrast, the function of PhLP is not as well understood; this proteinis even more widely expressed (Miles et al., 1993, Proc. Natl. Acad.Sci. USA 90, 10831-10835) and also binds the G protein beta/gammasubunits with high affinity (Schroder and Lohse, 1996, Proc. Natl. Acad.Sci. USA, 93, 2100-2104; Thibault et al., 1997, J. Biol. Chem., 272,12253-12256). It has been proposed that this protein represent aphosducin homologue that regulates a certain number ofG-protein-dependent pathways in many types of cells (Savage et al.,2000, J. Biol. Chem., Vol. 275, 39, 30399-30407).

However, no role of PhLP has been described to date in the regulation ofangiogenesis.

SRp75 belongs to the family of SR proteins due to the fact that itcontains in the N-terminal position a conserved domain RRM (RNArecognition motif), a glycine-rich region, an internal region homologouswith the RRM and a long (315 aa) C-terminal domain composed essentiallyof alternating serine and arginine residues (RS domain) (Zahler et al.,Mol. Cell. Biol. 1993 July; 13(7): 4023-8). The SR proteins constitute afamily of nuclear phosphoproteins which are necessary for constitutivesplicing but also influence the regulation of alternative splicing. TheSR proteins have a modular structure (one or two RRM domains and one RSdomain). Each domain in the SR proteins is a functional module. Thecoordinated action of the RRM domains determines their bindingspecificity to RNAs, whereas the RS domains function as spliceactivators (Caceres et al. 1997, J. Cell Biol., 139, 225-238; Chandleret al., 1997, Proc. Natl. Acad. Sci. USA, 94, 3596-3601; Mayeda et al.,1999, Mol. Cell. Biol., 19, 1853-1863; Graveley and Maniatis, 1998, Mol.Cell, 1, 765-771). Various studies have suggested the unique functionsin alternative splicing of the pre-mRNA for the particular SR proteins,especially since they are expressed differentially in a variety oftissues. These SR proteins are thus presented as crucial in theregulation of splicing during cell development and differentiation(Zahler et al., Science 1993 Apr. 9; 260 (5105): 219-222; Fu, 1993,Nature, 365 (6641): 82-8; Caceres and Krainer, 1997 (ed. Krainer),Oxford University Press, Oxford, UK, pp. 174-212; Valcarcel and Green,1996, Trends Biochem. Sci., 21(8): 296-301). A recent study showed avariable level of expression of SRp75 in different lymphoid cell lines(Dam et al., 1999, Biochim. Biophys. Acta; 1446(3): 317-33).

To date, no role in the regulation of angiogenesis has been describedfor either SFRS4 or SRp75 nor for the homologous protein of this factor.

Carboxypeptidase D (CPD of the S10 family of serine peptidases) is atransmembrane protein (180 kDa) which matures the proteins in thetrans-Golgi network and notably the proteins secreted via theconstitutive pathway such as the growth factors and their receptors:insulin receptor, insulin-like receptor of growth hormones (Reznik etal., 1998; J. Histochem. Biochem., 46, 1359). It is a carboxypeptidasewith an activity identical to that of carboxypeptidase E (CPElike) whichis widely distributed in the tissues. The carboxypeptidases intervene inthe elimination of basic amino acids from the C-terminal part of thepeptide to generate either the bioactive product or the precursor forthe formation of the C-terminal amide group (Fricker, 1988, Ann. Rev.Physiol., 50, 309-321; Fricker, 1991, (ed.) Peptide Biosynthesis andProcessing, pp. 199-230, CRC Press, Boca Raton, Fla.).

CPD is constituted in humans by three carboxypeptidase-like domains, ofone transmembrane domain and a small cytosol tail of 58 residues(Novikova et al., 1999, J. Biol. Chem., 274, 28887) capable of bindingthe phosphatase A protein (PP2A) (Varlamov et al., 2001, J. CellScience, 114, 311). This is a highly conserved protein among the specieswith similar enzymatic properties.

CPD is expressed to a high degree in the human placenta. It is foundnotably in the endothelial cells, the trophoblasts, the amnioticepithelial cells, the chorionic endothelial villus cells and the smoothmuscle vascular cells of umbilical cords (Reznik et al., 1998; J.Histochem. Cytochem., 46, 1359). CPE and CPD are also implicated in theproduction of the precursor of endothelin 1 (ET-1). This suggests thatCPE and CPD are implicated in the production of certain umbilical andplacental peptides having autocrine and/or paracrine functions.

To data there have been no descriptions of any regulatory role of theprotein CPD in angiogenesis.

The protein USP9X belongs to the family of UBPs (ubiquitin proteases), agroup of enzymes whose function is to invert the ubiquitination reactionby removing the ubiquitin residue from numerous substrates implicated incell division, growth, differentiation, signaling or activation oftranscription (Liu et al., 1999, Mol. Cell. Biol., 4, 3029-38; Zhu etal., 1996, Proc. Natl. Acad. Sci. USA 93: 3275-3279; Verma et al., 1995,Genes Dev. 9: 2723-2735). The ubiquitination of the proteins is animportant phenomenon in the regulation of the biological pathways suchas transduction activated by cytokines (Baek et al., 2001, Blood, 98,636-642). The UBPs are characterized by a conserved core domain withsurrounding divergent sequences and more particularly to the N-terminalpart enabling the specificity of the substrate. These N-terminaldivergences can alter the localization and confer multiple functions onthe different members of the large family of UBPs (Lin et al., 2001, J.Biol. Chem., 276(23): 20357-63). Certain specific proteases of ubiquitinhave already been described or suggested as implicated in certainbiological processes such as UBP109 in embryonic development (Park etal., 2000, Biochem. J., 349. 443-53), UBP43 in the differentiation ofhematopoietic cells (Liu et al., 1999, Mol. Cell. Biol., 4, 3029-38).The ubiquitination pathway is implicated in angiogenesis (Ravi et al.,2000, 14, 34-44; Sutter et al., Proc. Natl. Acad. Sci. USA, 97, Issue 9,4748-4753) but USP9X has not been described to date as a regulator ofangiogenesis.

The sequence of this mRNA has a coding sequence from nucleotide 136 tonucleotide 3795. There was thus identified a protein GS-P22 resultingfrom the translation of this mRNA. This protein nardilysine is composedof 1219 aa. It is identified as number SEQ ID No. 74 in the attachedsequence listing.

N-arginine dibasic convertase (NRD convertase; nardilysine; EC3.4.24.61) is a metalloendopeptidase of the family of the insulinasesthat cleave specifically the peptides (particularly the neuroendocrinepeptides such as somatosatin-28, dynorphine-A, the natriuretic atrialfactor) on the N-terminal side of an arginine residue at the level ofthe dibasic sites in vitro (Cohen et al., 1995, Methods Enzymol., 248,703-716; hospital et al., 1997, Biochem. J., 327, 773-779). Its exactfunction in vivo still remains poorly understood but many enzymes of thesame family are implicated in the maturation of the prohormones andproproteins (Winter et al., 2000, Biochem. J., 351, 755-764). The NRDconvertase activity is present principally in the endocrine tissues andto a majority degree in the testicles (Chesneau et al., 1994, J. Biol.Chem., 269, 2056-2061). It can be localized both in the cytoplasm and atthe cell surface (Hospital et al., 2000, Biochem. J., 349, 587-597).

At present, there is limited knowledge regarding the regulation of theexpression and activity of NRD convertase. The activity appears to beregulated by the amines that bind the acid domain (stretch) of theenzyme (Csuhai et al., 1998, Biochemistry, 37(11): 3787-94). It has alsobeen shown that retinoic acid can modulate the expression of the enzymein human neuroblast lines (Draoui et al., 1997, J. Neurooncol., 31,99-106).

In the adult rat, the regulated expression of NRD convertase duringspermatogenesis and its concentration in the flagellum suggests a roleof this enzyme in the differentiation of male germinal cells (Chesneauet al., 1996, J. Cell Sci. 109, 2737-2745). It has also been proposedthat this enzyme plays a specific role in neuronal development(Fumagelli et al., 1998, Genomics 47, 238-245). NRD convertase hasrecently been described as a new specific receptor of theheparin-binding EGF growth factor (HB-EGF) which controls cell migration(Nishi et al., 2001, EMBO J., 20(13): 3342-50).

In contrast, no role has been discovered to date for NRD1 in theregulation of angiogenesis.

The gene of acute lymphoblastic leukemia-1 (ALL)-1 or mycloid-lymphoidor mixed-lineage leukemia (MLL) or also designated human tri-thorax(HRX) on the human chromosome 11, band q23, is the site of many locallyregrouped chromosome alterations (deletions, partial duplications,translocations) associated with various types of leukemia. Thestructurally variant proteins derived from the altered gene are presumedto cause the malignant transformation of the precursor hematopoieticcells (Nilson, Br. J. Haematol., 1996, 93(4): 966-72; Kobayashi et al.,1995, Leuk. Lymphoma, 17(5-6): 391-9).

The protein MLL is a large nuclear protein with zinc finger motifs andSET domain, highly conserved, of 200 aa localized in the C-terminalpart. This protein is expressed to a high degree during embryogenesis;studies have shown that this protein is a positive regulator of thehomeobox genes Hox (Yu et al., 1995, Nature (London) 378, 505-508). Theprotein MLL is described as being implicated in transcriptionalmaintenance in the development which functions in multiple morphogeneticprocesses (Yu et al., 1998, Vol. 95, Issue 18, 10632-10636). It has beensuggested that the protein MLL plays a role in the regulation both ofcell proliferation and survival in the developing embryo (Hanson et al.,1999, Proc. Natl. Acad. Sci. USA, 96, Issue 25, 14372-14377).

To date, there have been no descriptions of any role of the protein MLLin the regulation of angiogenesis.

Since its identification in 1995, the gene ATRX synonyms XNP, XH2) hasbeen shown to be the gene of numerous forms of diseases; differentmental retardation syndromes associated with chromosome X are linked tothe mutations of this gene. (Review: Gibbons and Higgs, 2000, Am. J.Med. Genet., 97(3): 204-212.) This gene codes for a protein of thesubgroup SNF2 of the superfamily of the helicases and ATPases (Pickettset al., 1996, Hum. Mol. Genet. 5 (12): 1899-907); these domains suggestthat the protein ATRX has a role in transcriptional regulation via aneffect on the structure and/or the function of chromatin, but its exactrole still remains unknown. No role in the regulation of angiogenesishas been described to date.

The transporter of CMP-sialic acid is implicated in the process ofmaturation of glycosylation and more particularly of sialyation; itenables the translocation of cytosolic CMP-sialic acid through themembrane of the Golgi apparatus required for the sialyation of themembrane or secreted proteins as well as the lipids in this compartment.(Hirschberg and Snider, 1987, Ann. Rev. Biochem., 56, 63-88; Hirschberg,1996, in Organellar Ion channels and Transporters, Society of GeneralPhysiologists, 49^(th) Annual Symposium (Clapham, D. E. and Ehrlich, B.E., eds.), pp. 105-120, Rockefeller University Press, New York). Theregulation of the transport of CMP-sialic acid is still poorlyunderstood although an augmentation of sialyation was observed at thesurface of tumor cells (Santer et al., 1989, Eur. J. Biochem., 181,249-260; Saitoh et al., 1992, J. Biol. Chem., 267, 5700-5711; Bresalieret al., 1996, Gastroenterology, 110, 1354-1367; Gorelik et al., 1997,Cancer Res., 57, 332-336) and that the inhibition of the CMP-sialic acidtransporter reduces the growth and metastases of tumor cells (Harvey, B.E. and Thomas, P. (1993) Biochem. Biophys. Res. Commun. 190, 571-575).However, to date, no implication of this transporter in the regulationof angiogenesis has been reported.

Cbl-b belongs to the Cbl family, highly conserved among the species. TheCbl proteins are characterized in their N-terminal part by a putativedomain binding phosphotyrosines and a RING FINGER motif in theC-terminal part, the Cbl proteins of mammals containing a proline-richregion, conserved tyrosine residues and a zipper leucine motif. The Cblproteins participate in the signaling of the proteins of the tyrosinekinase receptors as well as the antigens and receptors of cytokines byassociating them at their cytoplasm tail providing the continuity of thesignal of these receptors. The protein Cbl is recruited by the tyrosinekinase module of these receptors and the phosphorylated tyrosines aftercell activation. Cbl functions as a docking protein and associatesitself with molecules containing the domains SH2 and SH3, including thefamily of the adapters Crk and Vav. It has been proposed that the Cblproteins are negative regulators of the signaling of the tyrosine kinasereceptors (Smit et al., Crit. Rev. Oncog. 1997; 8: 359-79) as well aspositive modulators of the signalization of receptors such as thesuperfamily of the TNF receptors (Arron et al., J. Biol. Chem. 2001 Aug.10; 276: 30011-7).

The protein Cbl-b, expressed at high levels in many tissues and cellsincluding the hematopoietic cells (Keane et al., Oncogene 1995 Jun. 15;10(12): 2367-77) is implicated in the installation of the lymphocyteactivation threshold (Bachmaier et al., Nature 2000, 403: 211-6; Fang etal., J. Biol. Chem. 2001; 276: 4872-8). The regulatory subunit P85 ofphosphatidylinositol 3-kinase (PI3K) was identified as being itssubstrate. Cbl-b, by its ligase activity of the protein ubiquitin E3,negatively regulates this regulatory subunit P85 (Fang D, Nat. Immunol.2001 September; 2(9): 870-5). Cbl-b is also a negative regulator of thesignalization of the receptor of the epidermal growth factor, EGFR(Ettenberg et al., Oncogene 1999; 18: 1855-66; Ettenberg et al., J.Biol. Chem. 20011 276: 27677-84).

In contrast, there have been no descriptions to date that Cbl-b plays arole in the regulation of angiogenesis.

The base chromatin unit in eukaryotes is the nucleosome. A nucleosome isconstituted by a 146-bp DNA sequence wound around an octamer ofproteins, the histones H2A, H2B, H3, H4 (Luger et al., 1997, Nature,389, 251-260). The heterogeneity in the structure of nucleosomes can bea transcriptional regulation mechanism. This heterogeneity is createdeither by post-translational modifications of the histones such asacetylation, phosphorylation, methylation, ubiquitination (Mizzen etal., Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 469-81; Workman andKinston, 1998, Ann. Rev. Biochem., 67: 545-579) or by the incorporationof histone variants in the nucleosome. The different histone variantsenable the specialization of the structure of the nucleosome forspecific purposes; the specific histone variants of sperm, for example,facilitate the dramatic compaction of DNA which occurs duringspennatogenesis (Wolffe, 1998, Chromatin: Structure and Function, 3^(rd)Ed., Academic Press, San Diego; Doenecke et al., 1997, Adv. Exp. Med.Biol., 424, 37-48).

The histone H2A.F/Z is a family of variants of the histone H2A which ishighly conserved across the species and substantially divergent from thehistone H2A of phase S in given species (Jackson et al., 1996, TrendsBiochem. Sci., 221, 466-467; Jiang et al., 1998, Biochem. Biophys. Res.Commun., 245, 613-617). The exact function of H2A.F/Z is still unknownbut this histone could play a role in transcriptional regulation becausein Tetrahymena its expression is associated with the transcriptionallyactive micronucleus and in Drosophila its incorporation in the chromatinduring development coincides with the beginning of the expression of thezygote gene (Clarkson et al., 1999, DNA Cell Biol., 18, 457-462;Stargell et al., 1993, Genes Dev., 7, 2641-2651).

In contrast, no role is known for the histone H2A.F/Z to date in theregulation of angiogenesis.

Casein kinase II (CKII) is a ubiquitous serine/threonine kinase which islocalized both in the cytosol as well as in the nucleus of eukaryotecells. CKII phosphorylates more than one hundred substrates, many ofwhich are implicated in the control of cell division and in thetransduction of the signal (review: Allende and Allende, 1995, The FASEBJournal, Vol. 9, 313-323). CKII exists in a tetramer form composed oftwo alpha and/or alpha′ catalytic subunits and two regulatory subunits(beta). The beta subunits appear to act so as to stabilize the alphaand/or alpha′ subunits and also influence the specificity of thesubstrate and the kinetic properties of the enzyme (Dobrowolska et al.,1999, Mol. Cell. Biochem., 191(1-2): 3-12). Certain studies have shownthat the activity of casein kinase is stimulated by growth hormones orfactors such as insulin, IGF-I, EGF (Sommercorn et al., 1987, Proc.Natl. Acad. Sci. USA, 84, 8834-8838; Klarlund et al., 1988, J. Biol.Chem., 263, 1872-1875; Ackerman and Osheroff, 1989, J. Biol. Chem., 264,11958-11965), this activation resulting from an augmentation of thephosphorylation of the beta subunit of casein kinase (Ackerman et al.,1990, Proc. Natl. Acad. Sci. USA, 87, 821-835). Various studies haveshown a deregulated expression of CKII in tumors. Recent studies havedemonstrated that the overexpression of CKII in tumor cells is notsolely a reflection of the proliferation of the tumor cells but also itcan reflect the pathobiological characteristics of the tumor. Thederegulation of CKII could influence the apoptotic activity of thesecells (review: Tawfic et al., 2001, Histol. Histopathol., 16(2):573-82). This enzyme is described as having a possible role inoncogenesis (Yu et al., J. Cell. Biochem., 1999, 74(1): 127-34).

In contrast, no differential expression of CKII or of its beta subunitduring angiogenesis has been described nor has a role in the regulationof angiogenesis been demonstrated to date.

Described recently, hemicentine is a protein of the extracellular matrixof the immunoglobulin superfamily which is implicated in cell attachmentand migration on the basal membrane (Vogel et Hedgecock, 2001,Development, 128(6): 883-94). Its role in the regulation of angiogenesishas not been described to date. This protein contains the sequence offibulin-6 which belongs to the fibulin family, proteins of theextracellular matrix and of the blood, the two members of which thathave been the most extensively studied are fibulin 1 and fibulin 2(Alexande and Detwiler, 1984, Biochem. J., 217, 67-71; Argraves et al.,1990, J. Cell Biol., 111, 3155-3164; Kluge et al., 1990, Eur. J.Biochem., 193, 651-659; Pan et al., 1993, J. Cell Biol., 123,1269-1277). They interact with the proteins implicated in cell adhesionsuch as fibronectin, laminin and fibrinogen (Brown et al., 1994, J. CellSci., 107 (Pt. 1), 329-38; Tran et al., 1995, J. Biol. Chem., 270(33):19458-64; Godyna et al., 1995, Matrix Biol., 14(6): 467-77) orendostatin which is an inhibitor of angiogenesis (Sasaki et al., 1998,EMBO J., 17(15): 4249-56) which confers on them a regulatory function invarious biological processes. Fibulin 1, for example, has been describedas possibly playing a role in the regulation of the neurotrophicactivity of the protein precursor amyloid beta (Ohsawa et al., 2001, J.Neurochem.; 76(5): 1411-20), in homeostasis and thrombosis (Tran et al.,1995, J. Biol. Chem., 270(33)L 19458-64).

In contrast, the function of fibulin 6 remains poorly understood and, inparticular, no role of this protein has been described to date in theregulation of angiogenesis.

The protein Syne-2 is poorly understood; it was recently described witha homologous protein, the protein Syne-1 (synaptic nuclear envelope-1).The protein Syne-1 is associated with the nuclear envelopes in the cellsof smooth, cardiac and skeletal muscle. Syn-1 is described as the firstprotein found to be associated selectively with the synaptic nucleus andit has been suggested that it is implicated in the formation ormaintenance of nuclear aggregates in the muscle junction (Appel et al.,2000, J. Biol. Chem., Vol. 275, Issue 41, 31986-31995). Syn-2 differsfrom Syn-1 in its distribution and in its level of expression which isweaker. The two homologous proteins exhibit repeated spectrin domainswhich are present in different proteins implicated in the structure ofthe cytoskeleton (Yan et al., Science 1993 Dec. 24; 262(5142): 2027-30).

No role of Syn-2 in the regulation of angiogenesis has been described todate.

The gene seladin-1 was recently identified in the human brain.Seladine-1 appears to be an important factor for the protection of cellsagainst the toxicity of the beta-amyloid peptide and oxidative stress(Greeve et al., 2000, J. Neuroscience, 20(19): 7345-7352). These authorssuggest that seladine-1 could be implicated in the regulation ofsurvival and cell death and that the diminished expression of thisprotein in specific neurons could be the cause for the selectivevulnerability in Alzheimer's disease.

In contrast, no role in the regulation of angiogenesis has beendescribed to date for seladine-1.

The protein CHD2 belongs to the family of CHD proteins characterized bya chromodomain. The “chromo” (CHRomatin Organization MOdifier) domain isa conserved region of circa 60 amino acids found in a variety ofproteins including the HP1 proteins of Drosophila melanogaster, which islinked to heterochromatin; 4 genes of these family have been identifiedin the human genome: CHD1, CHD2, CHD3 and CHD4 (Woodage et al., 1997,94, 11472-11477). Chromodomain confers on the protein a role in thecompaction of chromatin (Paro, 1990, Trends Genet. 6: 416-421; Singh etal., 1991, Nucleic Acids Res. 19: 789-794; Aasland and Stewart, 199,Nucleic Acids Res. 23: 3168-3173; Koonin et al., 1995, Nucleic AcidsRes. 23: 4229-4233; Messner et al., 1992, Genes Dev., 6, 1241-1254;James and Elgin, 1986, Mol. Cell. Biol. 6, 3862-3872). The CHDs containa second conserved domain, the domain Myb which is implicated in thebinding with DNA (Klempnauer and Sippel, 1987, EMBO J., 6: 2719-2725).In addition to these domains, CHD2 contains the domain SNF2, found inthe proteins implicated in a variety of processes such as the regulationof transcription (e.g.: SNF2, STH1, brahma, MOT1), repair of DNA (e.g.:ERCC6, RAD16, RAD5), recombination of DNA (e.g., RAD54) (review: Eisenet al., 1995, Nucleic Acids Res., 23(14): 2715-23) and lastly aconserved domain of the helicase superfamily, the “DEAH box helicases”.The helicases are implicated in the unwinding of nucleic acids (Matsonand Kaiser-Roger, 1990, Ann. Rev. Biochem. 59, 289-329). It was proposedthat the “DEAH box” helicases were implicated in the splicing of mRNAsand in the progression of the cell cycle (Ludgren et al., 1996, Mol.Biol. Cell 7, 1083-1094; Imamura et al., 1998, Nucleic Acids Res.,26(9): 2063-8).

The CHDs could play an important role in the regulation of thetranscription of genes (Delmas et al., 1993, Proc. Natl. Acad. Sci., 90,2414-2418; Woodage et al., 1997, Proc. Natl. Acad. Sci. USA, 94,11472-11477; Tran et al., 2000, The EMBO Journal, 19, 2323-2331).

A recent study showed that CHD4 is induced when endothelial cells arestimulated by TNF-alpha (Murakami et al., 2000, J. Atheroscler. Thromb.;7(1): 39-44).

No studies have demonstrated an implication of CHD2 in the regulation ofangiogenesis.

The role of the protein BRD2 is unknown. This protein is characterizedby two bromo domains. The bromo domain is a conserved region, firstidentified as a signature of 61-63 amino acids; its function beingunknown (Haynes et al., Nucl. Acids Res., 1992, 20, 2603). This domainwas subsequently identified in transcription factors, co-activators andother proteins are implicated in the transcription or remodeling ofchromatin and its boundaries were extended to 110 amino acids. Theincreasing number of proteins containing this domain is more than forty(Jeanmougin et al., 1997, Trends Biochem. Sci. 22, 151-153; Winston andAllis, 1999, Nature Struct. Biol. 6, 601-604). Certain proteins have asingle copy of the domain while others present two copies of the motifThe protein BRD2 is characterized by two bromo domains which is also thecase of the transcription factors BDFI (Tamkun, 1995, Curr. Opin. Genet.Dev., 5: 473-477) or the protein TAF11250, the largest subunit of themultiprotein complex TFIID implicated in the initiation of transcription(Jacobson et al., 2000, Science, 288(5470): 1422-5). The exact functionof this domain remains unknown but it is thought to be implicated inprotein-protein interactions and could be important for the assembly orthe activity of multiple complex components implicated in themodification of chromatin and the transcriptional control of a largevariety of eukaryote genes included those which control growth. A largevariety of functions directed on chromatin, including but not limited tophosphorylation, acetylation, methylation, co-activation ortranscriptional recruitment characterize the complexes which contain thebromo domains. Their versatility and ubiquity provides diverse, rapidand flexible transcriptional responses (review: Denis, 2001, Frontiersin Bioscience 6, d849-852).

The differential expression of proteins containing a bromo domain hasalready been demonstrated, notably that of a protein homologous to RING3kinase whose expression is induced by VEGF or bFGF in endothelial cells.It has been suggested that this protein is a new target of thesignalization pathway activated by VEGF and bFGF which enablesendothelial cells to enter into the proliferative phase of theangiogenesis process (BelAiba et al., 2001, Eur. J. Biochem., 268(16):4398-407).

In contrast, there have been no reports of BRD2 being implicated in theregulation of angiogenesis.

Syntaxin 3A belongs to the family of syntaxins/epimorphines which arecharacterized by a size between 30 and 40 kDa, a highly hydrophobicC-terminal end which is probably implicated in the anchoring of theprotein in the membrane and a well conserved central region whichappears to be in a coiled-coil conformation. The specific profile ofthis family is based on the most highly conserved region of thecoiled-coli domain. The syntaxins are implicated in the intracellulartransport of vesicles and their storage in the plasma membrane. Recentstudies suggest that different syntaxin isoforms could interact with adefined group of membrane transport proteins and thereby regulate theirtransport activity (review: Saxena et al., 2000, Curr. Opin. Nephrol.Hypertens., 9(5): 523-7).

Syntaxin 3A is one of the two isoforms (3A and 3B) identified in humans,stemming from an alternative splicing of the same gene. Augmentation ofthe expression of syntaxin 3A has already been demonstrated over thecourse of various biological processes such as the neutrophildifferentiation of HL-60 cells or in dentate granule cells during thepropagation of synaptic plasticity in the nervous system (Rodger et al.,1998, J. Neurochem., 71(2): 666-675; Martin-Martin et al., 1999, J.Leukoc. Biol., 65(3): 397-406).

In contrast, no increased expression of syntaxin has been reported todate in the course of angiogenesis and thus no implication in theregulation of angiogenesis.

Sharp (SMART/HDAC1 Associated Repressor Protein) is a recently isolatedgene (Nagase et al., 1999, DNA Res., 6(1): 63-70). SHARP is a potentialrepressor of transcription whose repression domain (RD) interactsdirectly with SMRT and at least 5 members of the NuRD complex(nucleosome remodeling and histone deacetylase activities) including thedeacetylase histones HDAC1 and HDAC2. SHARP moreover binds to the ARScoactivator of the RNA of the steroid receptor by an intrinsic domainbinding RNA and suppresses the transcription activity of the steroidreceptor. In this manner, SHARP has the capacity to modify both thebound and unbound nuclear receptors. The expression of SHARP is itselfinducible by steroids, suggesting a simple feedback mechanism for theattenuation of the hormonal response (Shi et al., Genes Dev. 2001 May 1;15(9): 1140-51. The deacetylase histones (HDAC) were shown to beimplicated in the induction of angiogenesis by suppressing theexpression of the tumor-suppressor genes (Kim et al., 2001, Nat. Med.,7(4): 437-43).

Nevertheless, no role of the SHARP protein has been described in theregulation of angiogenesis.

The exact role of the proliferation potential-related protein identifiedis still unknown. It presents as its homologous proteins zinc fingerdomains known to be implicated in protein-protein interactions. Thisprotein is homologous with a member of the family of theretinoblastoma-binding proteins (pRb), retinoblastoma-binding protein 6,also referred to as RBQ-1 (Sakai et al., 1995, Genomics, 30(1): 98-101).The protein pRb (suppressor of the retinoblastoma tumor) acts forcontrolling cell proliferation, inhibits apoptosis and induces celldifferentiation and does this by associating with a large number ofproteins (review: Morris and Dyson, 2001, Adv. Cancer Res.; 82: 1-54).

The proliferation potential-related protein has never been described todate as implicated in the regulation of angiogenesis.

The protein HIP1 (Huntingtin interacting protein 1) is a protein of 116kDa that binds the protein.

Huntingtin, product of the mutated gene in Huntington's disease(Kalchman et al., 1997, Nat. Genet., 16, 44-53; The Huntington's DiseaseCollaborative Research Group, 1993, Cell 72, 971-983). HIP1 ispredominantly expressed in the brain but is also detected in othertissues (Wanker et al., 1997, Human Molecular Genetics, Vol. 6,487-495). The function of HIP1 is not yet fully understood but it sharesthe biochemical characteristics and conserved domains with S1a2p, aprotein essential for the function of the cytoskeleton in Saccharomycescerevisiae (Kalchman et al., 1997, Nat. Genet. 16, 44-53; Holtzman etal., 1993, J. Cell Biol. 122, 635-644). HIP1 also contains a leucinezipper domain and is homologous in its C-terminal part with taline,cytoskeleton protein implicated in cell-substratum and cell-cellinteractions (Rees et al., 1990, Nature; 347(6294): 685-9). It hasrecently been shown that the protein HIP1 is implicated in apoptosis(Hackam et al., 2000, J. Biol. Chem., Vol. 275, Issue 52, 41299-41308).

No role of HIP1 in angiogenesis has been described to date.

Nucleoporin 88 (Nup88) is a protein of the nuclear membrane probablyimplicated in nucleocytoplasmic transport (Fomerod et al., 1997, EMBOJ., 16: 807-816; Fomerod et al., Oncogene 1966, 13: 1801-1808). Nup88was found to be associated with the central domain of CAN/Nup214, acomponent of the complex of a nuclear pore probably implicated in theimportation of nuclear proteins, the exportation of nuclear mRNAs andthe regulation of the cell cycle (van Deursen et al., 1996, EMBO J., 15:5574-5583). Nup88 has been shown to be widely distributed andoverexpressed in cancerous and fetal cells and tissues (Martinez et al.,1999, Cancer Research 59, 5408-5411; Gould et al., 2000, AmericanJournal of Pathology, 157: 1605-1613).

Nup88 to date has never been described as implicated in the regulationof angiogenesis.

The FKBPs (FK506 binding proteins) are major proteins binding theimmunosuppressive drug FK506 with high affinity in vertebrates (1990,Tropschug et al., Nature 346: 674-677; Stein, 1991, Curr. Biol. 1:234-236; Siekierka et al., 1990, J. Biol. Chem. 265: 21011-21015). Manymembers of the FKBP family have been identified in various tissues andvarious cellular compartments, the best known being FKBP12, a cytoplasmisoform (Galat and Metcalf, 1995, Prog. Biophys. Mol. Biol. 63, 67-118;Kay, 1996, Biochem. J. 314, 361-385). The FKBPs belong to a large familycis-trans peptidyl propyl isomerases (PPIase or rotamase). PPIase is anenzyme that accelerates the folding of the protein by catalyzing thecis-trans isomerization of the peptide bonds involving the prolineresidue (Fischer and Schmid, 1990, Biochemistry 29: 2205-2212). TheFKBPs are known to be implicated in many cellular processes such ascellular signaling, protein transport and transcription. Studies ofinterruption of the gene coding for the FKBPs in plants and animals haveemphasized the importance of this family of proteins in the regulationof cell division and differentiation (review: Harrar et al., 2001 TrendsPlant Sci.; 6: 426-331). However, despite the fact that they bindsurface receptors and despite their Ppiase activity, their physiologicalfunction has yet to be defined. It was recently proposed that thePpiases play a role in the functional rearrangement of the components inthe heterocomplex receptors (Schiene-Fisher and Yu, 2001, FEBS Lett.,495(1-2): 1-6).

It has not been claimed to date that the FKBP proteins are implicated inthe regulation of angiogenesis.

The complementary DNA (cDNA) of the SALF protein (stonedB/TFIIA-alpha/beta-like factor) was recently identified from a bank ofcDNA originating from the human placenta (Ashok et al., 1999, J. Biol.Chem., Vol. 274, Issue 25, 18040-18048). The role of SALF has yet to beidentified but this cDNA, characterized as rare, is identical to thesequence of the ALF protein with a more extended N-terminal sequencecontaining a domain homologous to the Stone B protein of Drosophila(Andrewe et al., 1996, Genetics 143, 1699-1711) and to the adaptorproteins of Clathrina, μ ₁ (AP47) and μ₂ (AP50) (Thurieau et al., 1988,DNA (NY) 7, 663-669; Nakayama et al., 1991, Eur. J. Biochem. 202,569-674). The protein ALF (TFIIA-alpha/beta-like factor) is also a newfactor which is a functional homolog of the transcription factor TFIIAalpha/beta which, with the factor TFIIA gamma, can stabilize theinteractions of the element TBP (TATA-binding protein)-TATA and maintainthe in vitro transcription dependent of RNA polymerase II. The proteinALF is described as a general transcription factor specific of thetesticles (Ashok et al., 1999, J. Biol. Chem. Vol. 274, Issue 25,18040-180048).

The domain of Stoned B/clathrin AP-like homology of SALF also reveals apotential function in the dependent transport of clathrin in themembrane proteins.

Chen et al. (2001, Biochem. Biophys. Res. Commun. 23; 281(2): 475-482)showed that the gene of the factor SALF is induced by retinoic acid (orthe retinoids) in cultured smooth muscle cells.

In contrast, no implication of SALF in the regulation of angiogenesishas been reported to date.

The recently described protein P29 interacts with the protein GCIP(Grap2 cyclin-D interacting protein), itself interacting with cyclin Dand the protein Grap2 (Chang et al., Biochem. Biophys. Res. Commun. 2000Dec. 20; 279(2): 732-7). Grap2 is an adaptor protein specific of theleukocytes of the immune tissues (Qiu et al., 1998, Biochem. Biophys.Res. Commun. 253, 443-447). The adaptor proteins play an essential rolein the formation of intracellular signalization complexes relaying theextracellular signals from the plasma membrane to the nucleus of thecell. Grap2 is a central linking protein in the signalization andactivation of the immune cells. The type D cyclins respond to theextracellular mitogenic signals (Sherr, 1993, Cell 73, 1059-1065). Theprotein GCIP, which is expressed in a ubiquitous manner in all humantissues, interacts with Grap2 and the D cyclins. Its expressionregulates the phosphorylation of the retinoblastoma (Rb) protein andthereby the transcription pathway controlled by the transcription factorE2F1 belonging to a family of factors that play a major role in theproliferation, differentiation, apoptosis and progression of the cellcycle (Nevins, 1998, Cell Growth Differ. 9, 585-593; Chellappan et al.,1998, Curr. Top. Microbiol. Immunol. 227, 57-103; Dyson, 1998, GenesDev. 12, 2245-2262). It has therefore been suggested that GCIP plays arole in the control of cell proliferation and differentiation via thesignalization pathways controlled by the Grap2 and cyclin D proteins(Xia et al., 2000, J. Biol. Chem., 275(27): 20942-8). The protein P29which has been localized in the nucleus and is present in multipletissues has been found to be associated with GCIP and therefore could beimplicated in the functional regulation of GCIP (Chang et al., Biochem.Biophys. Res. Commun. 2000 Dec. 20; 279(2): 732-7).

The protein P29 thus appears to play a role in the signaling pathwaysimplicated in cell proliferation and differentiation. However no role inthe regulation of angiogenesis has been demonstrated to date either forthe protein P29 or for the identified homologous protein.

The gene of TMEM2 has recently been described (Scott et al., Gene 2000;246(1-2): 265-74); based on its structure, it was suggested that thecoded protein TMEM2 was transmembranous. The presence of the RGD motifsuggesting a role in cellular adhesion is an implication in the cellularsignalization pathways.

No role has been described to date for this protein and in particular norole in the regulation of angiogenesis has been demonstrated.

Although few studies to date have focused on the protein called Dorfin,its gene was recently identified. This gene is ubiquitously expressed inmany organs. It binds specifically to the conjugating ubiquitin enzymes,UbCH7 and UbCH8, via its RING-FINGER/IBR domain. Dorfin is proposed as anew member of the RING Finger type ubiquitin ligases. This protein islocalized in the centrosome and probably acts in the organizationcenters of the microtubules (Niwa et al., 2001, Biochem. Biophys. Res.Commun., 281(3): 706-13). No differential expression of Dorfin nor arole in the regulation of angiogenesis have been reported to date.

The protein TM4SF2 belongs to the type 4 transmembranous superfamilycertain of whose members are known to be implicated in angiogenesis suchas CD9 and PETA-3/CD151 (Klein-Soyer et al., 2000, Arterioscler. Thromb.Vasc. Biol., 20: 360-9; Sincock et al., 1999, J. Cell. Science, 112,833-844). In contrast the function of TM4SF2 remains unknown. Thisprotein is homologous with the protein TALLA-1, proposed as a specificsurface marker of neuroblastomas and neuroblastic leukemia (Takagi etal., Int. J. Cancer, 1995, 61(5): 706-15).

No role for the protein TM4SF2 in angiogenesis has been reported todate.

The ecto-ATPases (ATPases of the cell surface) are enzymes that areubiquitous in the cells that hydrolyze extracellular ATP and ADP intoAMP (review: Plesner, 1995, Int. Rev. Cytol., 158: 141-214). Thepresence of the ATPDases was demonstrated in the aortic endothelialcells and the smooth muscle cells and described as possibly playing aregulatory role in homeostasis and platelet reactivity by hydrolyzingATP and ADP (Robson et al., 1997, J. Exp. Med., 185(1): 153-163).Vascular ATPDase is closely homologous to the glycoprotein CD39 whoseaccession number in the GENBANK database is S73813 and which isidentified by the number SEQ ID No. 290 in the attached sequencelisting, activation antigen of lymphoid cells, also expressed by humanendothelial cells (Kaczmarek et al., 1996, J. Biol. Chem., 271, 51,33116-33122). Recently, Goepfert et al. (2001, Circulation, no. 104(25): 3109-3115), implanting bodies containing Matrigel in mutant micecharacterized by deficiency of expression of CD39, showed the absence offormation of neovessels around the implanted bodies. These observationsled the authors to suspect a role for CD39 in the angiogenesisphenomenon. However, the experimentation described supports a poorlydefined role of CD39 in angiogenesis rather than a direct andincontestable role. In fact, due to the fact that mutant mice deficientin CD39 exist, it is possible that embryos of CD39-deficient mice coulddevelop. However, without angiogenesis there is no possible embryonicdevelopment. Consequently, either CD39 has a role in angiogenesis andtherefore there would not exist viable CD39-deficient mutant mice, orCD39 has no role in angiogenesis. In another article, Goepfert et al.(2001, Circulation, 104 (25): 3109-15) did not demonstrate the absenceof expression of CD39 in the mutant mouse endothelial cells employed.

In sum, the study by Goepfert et al. (2001, Circulation, 104(25):31309-15) does not provide proof of any role of CD39 in angiogenesis.

Control of angiogenesis thus represents a strategic axis, both for basicresearch (to improve our comprehension of numerous pathologicalphenomena linked to angiogenesis) and for the development of newtherapies intended for treating pathologies linked to angiogenesis.

To control angiogenesis, many pharmaceutical groups have developedtherapeutic strategies based directly on the use of paracrine signals asstimulatory and inhibitory factors as agents for promoting or inhibitingangiogenesis. These strategies are based essentially on the use of thesefactors in their polypeptide form as stimulatory or inhibitory agents ofangiogenesis, or more recently in the form of expression vectors codingfor the selected factors.

SUMMARY

This disclosure relates to a pharmaceutical composition including apharmaceutically acceptable carrier and an at least one active agentselected from the group consisting of (i) a nucleic acid molecule of agene of an endothelial cell, the expression of which is induced by anangiogenic factor and inhibited by an angiostatic agent; (ii) acomplementary sequence or a fragment or derivative of the nucleic acidmolecule of (i); (iii) a polypeptide sequence coded by the nucleic acidmolecule of (i) or (ii); (iv) an antisense nucleic acid molecule thatinhibits the expression of a nucleic acid molecule according to (i); andan antibody that binds to a polypeptide sequence according to (ii).

This disclosure also relates to an antibody that has an affinity for oneof the polypeptide sequences identified by SEQ ID No. 54 to SEQ ID No.102 or SEQ ID No. 291 to SEQ ID No. 297, or fragments or derivativesthereof.

This disclosure further relates to a method for the preparation of anantibody that has an affinity for one of the polypeptide sequencesidentified by SEQ ID No. 54 to SEQ ID No. 102 or SEQ ID No. 291 to SEQID No. 297, or fragments thereof, including immunizing animmunocompetent cell of an animal in vivo or in vitro with at least onepolypeptide sequence identified by SEQ ID No. 54 to SEQ ID No. 102 orSEQ ID No. 291 to SEQ ID No. 297.

This disclosure still further relates to a pharmaceutical compositionincluding one or more antibodies that have an affinity for one of thepolypeptide sequences identified by SEQ ID No. 54 to SEQ ID No. 102 orSEQ ID No. 291 to SEQ ID No. 297, or fragments or derivatives thereofand a pharmaceutically acceptable carrier.

This disclosure further still relates to an antisense nucleotidesequence including at least 10 nucleotides selected from the set ofsequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQID No. 225 and SEQ ID No. 284 to SEQ ID No. 288.

This disclosure also further relates to a mammalian expression vectorincluding at least one antisense sequence.

This disclosure again relates to a mammalian expression vector includingat least one nucleotide sequence selected from among the set ofsequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQID No. 225 and SEQ ID No. 284 to SEQ ID No. 290.

This disclosure yet again relates to a method for the preparation of agenetically modified cell underexpressing a gene implicated in anangiogenic disorder, comprising inserting into a mammal cell a vectorincluding at lest one antisense sequence.

This disclosure also again relates to a genetically modified cell thatoverexpresses at least one gene implicated in angiogenesis, wherein theat least one gene is selected from among the set of sequences identifiedby the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ IDNo. 284 to SEQ ID No. 290, or fragments or derivatives thereof.

This disclosure further again relates to a method for the diagnosis orprognosis of an angiogenic pathology in a mammal, comprising the stepsof detecting in vitro in the cells of the mammal the overexpression orunderexpression of one or more nucleotide sequences selected from theset identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No.225 and SEQ ID No. 284 to SEQ ID No. 290.

This disclosure still further again relates to a method for thediagnosis or prognosis of an angiogenic pathology in a mammal,comprising of detecting in vitro in the cells of the mammal theoverexpression or underexpression of one or more polypeptide sequencesidentified by the numbers SEQ ID No. 54 to SEQ ID No. 102, or SEQ ID No.291 to SEQ ID No. 297.

This disclosure also further again relates to a method for theverification of the therapeutic efficacy of an angiogenic treatment in amammal, comprising identifying in vitro in a cell population of themammal the overexpression or underexpression of at least one geneimplicated in an angiogenic disorder identified by the numbers SEQ IDNo. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No.290.

This disclosure also yet again relates to a method for screening forcompounds useful in the treatment of an angiogenic disorder of a mammal,comprising the steps of a) detecting expression of one or morenucleotide sequences selected from the set identified by SEQ ID No. 1 toSEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in afirst mammalian cell population contacted with a test compound; b)detecting expression of one or more of the nucleotide sequences in asecond reference cell population whose angiogenic state is known; and c)identifying differences in the level of expression of one or more of thenucleotide sequences in the first and second cell populations, whereindifferences in expression of the nucleotide sequences indicates the testcompound has a therapeutic effect on an angiogenic disorder.

This disclosure then also relates to a device including a support,wherein the support comprises one or more specific probes of one or morenucleotide sequences selected from the set identified by the numbers SEQID No. 1 to SEQ D No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ IDNo. 290, or fragments or derivatives thereof.

This disclosure then again relates to a kit for the measurement of thedifferential display of genes implicated in angiogenic disorders,including a device including a support, specific primers and accessoriesrequired for (i) amplification of nucleotide sequences extracted from asample; (ii) hybridization of the amplified nucleotide sequences withprobes of the device; and (iii) performance of differential displaymeasurements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 1A) GS-V1; 1B) GS-V2; 1C) GS-V4; 1D) GS-V5; 1E) GS-V15; and 1F)empty (control) vector.

FIG. 2 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 2A) GS-V3; 2B) GS-V14; and 2C) empty (control) vector.

FIG. 3 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 3A) GS-V6; 3B) GS-V8; 3C) GS-V10; and 3D) empty (control)vector.

FIG. 4 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 4A) GS-V7; 4B) GS-V9; 4C) GS-V12; 4D) GS-V12; 4E) GS-V14 and 4F)empty (control) vector.

FIG. 5 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 5A) GS-V16; 5B) GS-V17; 5C) GS-V18; 5D) GS-V19; 5E) GS-V21 and5F) empty (control) vector.

FIG. 6 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 6A) GS-V22; 6B) GS-V24; 6C) GS-V25; 6D) GS-V26; 6E) GS-V27; and6F) empty (control) vector.

FIG. 7 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 7A) GS-V28; 7B) GS-V29; 7C) GS-V30; 7D) GS-V31; 7E) GS-V32; and7F) empty (control) vector.

FIG. 8 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 8A) GS-V33; 8B) GS-V34; 8C) GS-V35; 8D) GS-V37; 8E) GS-V38; and8F) empty (control) vector.

FIG. 9 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 9A) GS-V40; 9B) GS-V42; 9C) GS-V43; 9D) GS-V44; 9E) GS-V45; and9F) empty (control) vector.

FIG. 10 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 10A) GS-V20; 10B) GS-V23; 10C) GS-V36; 10D) GS-V39; 10E) GS-V41;and 10F) empty (control) vector.

FIG. 11 is a photomicrograph of a human endothelial cell culture showingcapillary tube formation upon transfection of cells with expressionvectors 11A) GS-V46 and 11B) empty (control) vector.

DETAILED DESCRIPTION

A method for the identification of new genes implicated in theregulation of angiogenesis has been developed. This method was theobject of a French patent application published as FR no. 2798674 and ofan International patent application published as WO 01/218312, theentire disclosures of which are herein incorporated by reference. Thismethod has the distinctive characteristic of faithfully translating theinnermost mechanisms regulating angiogenesis, taking into account all ofthe extracellular factors described as regulatory agents ofangiogenesis; i.e., the angiogenic factors and angiostatic factors aswell as the different components of the extracellular matrix. Thismethod consists of bringing to bear these different extracellularfactors via four clearly defined experimental conditions, in whichendothelial cells are cultured on a component and/or on a clearlydefined mixture of multiple components of the extracellular matrix andplaced under the four experimental conditions, i.e.:

-   -   A control condition in which the endothelial cells are not        stimulated.    -   An angiogenic condition in which the endothelial cells are        stimulated by one or more angiogenic factors.    -   An angiogenesis inhibition condition in which the endothelial        cells are stimulated by one or more angiogenic factors and        brought into the presence of one or more angiostatic conditions.    -   Another control condition in which the endothelial cells are        stimulated by one or more angiostatic factors.

By means of these four conditions, it is possible to obtain mRNApreparations specific of angiogenesis, i.e., of the angiogenic stateand/or the inhibition of angiogenesis, and to make it possible to detectgenes coding for the cellular constituents implicated in the regulationof angiogenesis, including positive regulators and negative regulators.Thus, the method described above enables the systematic screening of allof the angiogenic and angiostatic factors, as well as the differentcomponents of the extracellular matrix, for the purpose of revealing andidentifying the genes coding for the cellular constituents implicated inthe regulation of angiogenesis. Moreover, given that the gene expressioncan be analyzed all along the pathway of the formation of neovessels byendothelial cells, this approach constitutes an in vitro methodologymaking it possible to link the gene expression with the biologicalfunctional parameters of angiogenesis.

The identification of the fifty-four genes presented below wasimplemented by means of the methodology described above, using theangiogenic and angiostatic factors, as well as type I collagen ascomponent of the extracellular matrix for reproducing the fourexperimental conditions.

The fifty-four new genes identified by the sequences SEQ ID No. 1 to SEQID No.: 53 and SEQ ID No. 225 in the attached sequence listing areimplicated in the regulation mechanism of angiogenesis.

The disclosure also provides a pharmaceutical composition active forinhibiting angiogenesis, comprising a pharmaceutically acceptablecarrier and, as active agent, at least one substance selected fromamong: (i) a nucleic acid molecule of a gene of an endothelial cell, theexpression of which is induced by an angiogenic factor and inhibited byan angiostatic agent, or a complementary sequence or a fragment orderivative thereof; (ii) a polypeptide sequence coded by the nucleicacid molecule or fragment or complement thereof; (iii) a moleculecapable of inhibiting the expression of a nucleic acid moleculeaccording to (i) or which binds to a polypeptide sequence according to(ii). Pharmaceutical compositions can be for human or veterinary use,and are preferably sterile and pyrogen free. Pharmaceutical compositionscomprise, in addition to at least one active ingredient, at least onepharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include water (e.g., sterile water for injection);saline solutions such as physiological saline or phosphate bufferedsaline (PBS); polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose; stabilizing orpreservative agents, such as sodium bisulfite, sodium sulfite andascorbic acid, citric acid and its salts, ethylenediaminetetraaceticacid, benzalkonium chloride, methyl- or propylparaben chlorobutanol; andcombinations thereof.

According to one aspect, the pharmaceutical composition comprises as anactive ingredient at least one nucleotide sequence selected from the setof nucleotide sequences identified as numbers SEQ ID No. 1 to SEQ ID No.53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attachedsequence listing, their complementary sequences and their correspondingantisense sequences, or one of their fragments or derivatives.

In the context of this disclosure, the following should be considered tobe equivalent sequences (also called “derivative sequences” or“derivatives”): nucleotide sequences presenting minor structuralmodifications not changing their function, such as deletions, mutationsor additions of bases, the identity of which is at least 90% with thenucleotide sequences identified under the numbers SEQ ID No.: 1 to SEQID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in theattached sequence listing. One skilled in the art can readily identifyderivatives of the present nucleic acids by testing them for the abilityto regulate angiogenesis in the human endothelial cell culture assaysdescribed in the “Examples” section below. As used herein, “fragments”of the present nucleic acids comprise a smaller, contiguous sequence ofnucleotides found within a larger nucleic acid sequence.

According to another aspect, the angiogenesis regulatory pharmaceuticalcomposition comprises at least one angiogenesis inhibitory sequence.

According to one aspect, the angiogenesis regulatory pharmaceuticalcomposition comprises at least one angiogenesis stimulatory sequence.

According to one aspect, the pharmaceutical composition comprises one ormore angiogenesis inhibitory sequences comprising an antisense sequencecomprising all or part of at least one sequence selected from among SEQID No. 1 to SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 toSEQ ID No. 15, SEQ ID No. 17 to SEQ ID No. 53, SEQ ID No. 225 and SEQ IDNo. 284 to SEQ ID No. 290 in the attached listing of sequences.

The pharmaceutical composition preferably comprises one or moreantisense sequences selected from among SEQ ID No. 103 to SEQ ID No.107, SEQ ID No. 109, SEQ ID No. 111 and SEQ ID No. 113 to SEQ ID No. 148in the attached sequence listing.

According to a second aspect, the pharmaceutical composition comprisesone or more stimulatory sequences of angiogenesis, comprising anantisense sequence of at least one of the sequences SEQ ID No. 6, SEQ IDNo. 8, SEQ ID No. 10 and SEQ ID No. 16 in the attached sequence listing.

The pharmaceutical composition preferably comprises antisense sequencesselected from among sequences SEQ ID No. 108, SEQ ID No. 110 and SEQ IDNo. 112 in the attached sequence listing.

We also provide a pharmaceutical composition intended for the diagnosisand/or treatment of pathologies linked to angiogenesis, characterized inthat the pharmaceutical composition contains at least one polypeptidesequence selected from among the polypeptide sequences identified by thenumbers SEQ ID No. 54 to SEQ ID No. 102 or among the polypeptidesequences identified by the numbers SEQ ID No. 291 to SEQ ID No. 297 inthe attached sequence listing.

In the context of this disclosure, equivalent sequences (also called“derivative sequences” or “derivatives”) should be considered to bethose polypeptide sequences presenting minor structural modificationsnot changing their function, such as deletions, mutations or additionsof amino acid residues, the identity of which is at least 85%,preferably at least 90%, with the polypeptide sequences identified bythe numbers SEQ ID No. 54 to SEQ ID No. 102 or with the polypeptidesequences identified by the numbers SEQ ID No. 291 to SEQ ID No. 297 inthe attached sequence listing. One skilled in the art can readilyidentify derivatives of the present polypeptides by testing them for theability to regulate angiogenesis in the human endothelial cell cultureassays described in the “Examples” section below. As used herein,“fragments” of the present polypeptides comprise a smaller, contiguoussequence of amino acids found within a larger polypeptide sequence.

We also provide a pharmaceutical composition intended for the diagnosisand/or treatment of pathologies linked to angiogenesis, comprising atleast one antagonist of one or more of the above-mentioned polypeptidesequences, and a pharmaceutically acceptable carrier.

As used herein, the term “antagonist” is understood to mean any compoundwhich inhibits the biological activity of the polypeptide sequences inthe mechanism of angiogenesis.

For example, a suitable antagonist can comprise an antibody having anaffinity for a polypeptide sequence.

We also provide antibodies having an affinity for each of thepolypeptide sequences identified by the numbers SEQ ID No. 54 to SEQ IDNo. 102, or for the polypeptide sequences identified by the numbers SEQID No. 291 to SEQ ID No. 297 in the attached sequence listing, as wellas the therapeutic compositions containing such antibodies.

Antibodies can be obtained by any in vivo or in vitro immunizationmethod from an animal, notably a vertebrate and preferably a mammal,with any one of the polypeptide sequences identified by the numbers SEQID No. 54 to SEQ ID No. 102, or with the polypeptide sequencesidentified by the numbers SEQ ID No. 291 to SEQ ID No. 297 in theattached sequence listing, or one of their fragments which induceimmunogenicity to the protein. Suitable immunization methods that can beused to produce antibodies are within the skill in the art; see, e.g.,Kohler G. and Milstein C., Nature 1975 Aug. 7; 256(5517): 495-497, theentire disclosure of which is herein incorporated by reference.

The antibodies can be polyclonal or monoclonal antibodies.

We also provide a therapeutic or diagnostic composition comprising oneor more antibodies having an affinity for one or more of the polypeptidesequences identified by the numbers SEQ ID No. 54 to SEQ ID No. 102 orfor the polypeptide sequences identified by the numbers SEQ ID No. 291to SEQ ID No. 297, or for one of their fragments or derivatives, whichinduce immunogenicity to the protein prepared as indicated above.

Another object of this disclosure pertains to antisense nucleotidesequences of the nucleotide sequences identified by the numbers SEQ IDNo. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No.290 in the attached sequence listing.

In the context of this disclosure, the term “antisense sequence” isunderstood to mean any DNA sequence of at least 10 nucleotidescomplementary to at least a portion of an mRNA, which inhibits itsexpression of that mRNA; i.e., its translation into a protein.

For example, the antisense sequences can have an identity of at leastabout 80%, at least about 85% or at least about 90%, preferably at leastabout 95%, and more preferably at least about 99%, with a sequenceselected from among the sequences identified by numbers SEQ ID No. 103to SEQ ID No. 148 in the attached sequence listing.

We also provide a mammalian expression vector comprising at least oneantisense sequence as defined above.

The vector can be selected from among the group of vectors GS-V1 toGS-V46 carrying the at least one of SEQ ID No. 149 to SEQ ID No. 194 inthe attached sequence listing.

The introduction of the sequences SEQ ID No. 103 to SEQ ID No. 148 intomammalian expression vectors and the subsequent insertion of the vectorsinto mammalian cells produces cell lines underexpressing the genesintervening in the mechanism of angiogenesis.

We also provide a mammalian expression vector, the vector comprising atleast one antisense sequence of at least one of the sequences identifiedby the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ IDNo. 284 to SEQ ID No. 290 in the attached sequence listing, as well as apromoter which enables the expression of the antisense DNA.

Specific primers for each of the identified sequences are designed forthe construction of these vectors.

These particularly preferred primers are indicated in Table I below andidentified by the sequence numbers SEQ ID No. 195 to SEQ ID No. 222, SEQID No. 226 to SEQ ID No. 283 and SEQ ID No. 298 to SEQ ID No. 299 in theattached sequence listing.

Amplification of the bacterial plasmid comprising the cloned gene isadvantageously effected by means of primers hybridizing with the regionsof the plasmid surrounding the cloned gene. The primers also comprise ontheir ends certain restriction sites not contained in the clonedfragment or present in the multisite region of the expression vector.

For example, in the context of the cloned fragments employed, therestriction sites employed with the expression vector pCI are the sitesSalI and MluI. These two restriction sites can be interchanged dependingon whether the fragment was cloned in the bacterial plasmid in its senseor antisense orientation.

As an example, the primers GS-PGS-F (SEQ ID No. 223) and GS-PGM-R (SEQID No. 224) are used for fragments cloned in the sense orientation inthe bacterial plasmid (GS-N15).

These particular primers can be used in a universal manner fortransferring all of the fragments cloned in sense orientation in abacterial vector for integrating the expression vector in the antisenseorientation.

The primers GS-PGM-F (SEQ D No. 300) and GS-PGS-R (SEQ ID No. 301) areused for fragments cloned in antisense orientation in the bacterialplasmid (GS-N46).

These particular primers can be used in a universal manner fortransferring all of the fragments cloned in antisense orientation in abacterial vector for integrating the expression vector in the antisenseorientation.

We also provide a mammalian expression vector comprising at least onenucleotide sequence selected from among the set of sequences identifiedby the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ IDNo. 284 to SEQ ID No. 290 in the attached sequence listing, or one oftheir fragments or derivatives.

These vectors are useful for preparing pharmaceutical compositionsintended for the treatment of angiogenic disorders, for verifying theefficacy of a treatment of an angiogenic disorder in a mammal, notably ahuman being, or for verifying the functionality of genes possiblyimplicated in the mechanism of angiogenesis in a mammal.

We therefore also provide a genetically modified cell comprising atleast one of the vectors comprising the antisense sequences forinhibiting expression of at least one nucleotide sequence selected fromamong the sequences SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 andSEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing.

We also provide a method for the preparation of a genetically modifiedcell line expressing a nucleotide sequence in a stable manner, thevector comprising at least one antisense sequence of at least one of thesequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequencelisting, as well as a promoter which enables the expression of theantisense DNA. The method comprises the following steps:

a) introducing a gene of resistance to at least one antibiotic into thegenetically modified cell;

b) culturing the cells obtained in step (a) in the presence of theantibiotic; and

c) selecting the viable cells.

We also provides a pharmaceutical composition intended for the diagnosisand/or treatment of pathologies linked to angiogenesis comprising asactive principle the genetically modified cell.

We also provide a genetically modified cell comprising at least onevector comprising a nucleotide sequence selected from among the set ofsequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ DNo. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequencelisting, or one of their fragments or derivatives.

This disclosure thus pertains to a method for the preparation of a lineof genetically modified cells expressing a nucleotide sequence in astable manner, the vector comprising at least one of the sequencesidentified by the numbers SEQ. ID No. 1 to SEQ ID No. 53, SEQ ID No. 225and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing,or one of their fragments or derivatives, as well as a promoter enablingthe expression of the antisense DNA. The method comprises the followingsteps:

a) introducing a gene of resistance to at least one antibiotic into thegenetically modified cell;

b) culturing the cells obtained in step (a) in the presence of theantibiotic; and

c) selecting the viable cells.

It is thus possible to isolate human cells and transfecting them invitro with at least one of the vectors defined above, which vectors codefor at least one of the genes whose sequences are identified by thenumbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284to SEQ ID No. 290, or one of their fragments or derivatives. Thesegenetically modified cells can then be administered to a mammal,preferably a human being.

Therapeutic compositions containing such cells can be presented in theform of simple cellular suspensions, but can also be encapsulated in asuitable device using, e.g., semipermeable membranes.

Another object of this disclosure is a method for the preparation of aprotein coded by at least one of the nucleic acids whose sequences areidentified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing,or one of their fragments or derivatives.

These proteins, which are identified by the sequences SEQ ID No. 54 toSEQ ID No. 102 and SEQ ID No. 291 to SEQ ID No. 297 in the attachedsequence listing, or their fragments or derivatives can be produced invitro in the form of recombinant proteins by introducing into a suitablehost a corresponding suitable expression vector. The proteins, orfragments or derivatives thereof, thus produced are then purified andsubsequently used as a therapeutic agent.

A method for preparing a recombinant protein comprises the steps of:

-   -   a) constructing an expression vector comprising at least one        sequence from among those identified by the numbers SEQ ID No. 1        to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID        No. 290 in the attached sequence listing, or one of their        fragments or derivatives;    -   b) introducing the vector into a cellular host;    -   c) culturing the cells in a suitable medium; and    -   d) purifying the expressed proteins or one of their fragments or        derivatives.

We also provide a recombinant protein obtained by the above-describedmethod.

As an example, systems for expressing recombinant proteins in bacteriasuch as E. coli can be used for expressing proteins (includingnon-glycosylated proteins).

The entire or partial sequence of the nucleic acid of interest can beamplified by PCR using specific primers, which preferably containdifferent restriction enzyme digestion sites at the ends so as to enableorientation of the gene in the expression vector. The amplified DNA ispurified, then digested by the appropriate restriction enzymes. Thedigested nucleic acid is then inserted by standard ligation techniquesin the expression vector previously digested by these same restrictionenzymes. Any suitable vector can be used, such as the vector pBR322(Bolivar et al., Gene 2 (1977) 95-113) or its derivatives containing theRNA polymerase promoter of the bacteriophage T7 for a high level ofexpression. Such pBR322 derivatives include the plasmid pET3a (Studierand Moffatt, 1986, J. Mol. Biol., 189(1): 113-30).

Preferably, vectors used contain sequences that code for selectionmarkers (resistance to antibiotics), a multiple cloning site containingthe sites of restriction enzymes suitable for the insertion of DNA, andthe cell/host system is preferably an inducible system such as that usedfor the in vivo radiotagging of the growth factor FGF2 (Colin et al.,1997, Eur. J. Biochem., 249, 473-480) and already described by Patry etal. (1994, FEBS Lett., 349(1): 23-8), the disclosures of which areherein incorporated by reference. Suitable vectors can also contain aregion coding for a polyhistidine tail at the end of the protein ofinterest to facilitate purification of encoded proteins.

In the practice of the present methods, the amplified DNA is ligated inthe plasmid which is transformed in the bacterium according to anysuitable method, such as the method described by Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The transformed cells canthen be spread on agar LB medium containing antibiotics. Coloniesresistant to the antibiotics, which are formed by bacteria carrying therecombinant plasmid, are isolated. The plasmid DNA can then be isolatedfrom the bacteria and sequenced to confirm the construction of thevector. The production and purification of the recombinant protein fromthe isolated vectors can be performed as described ((Patry et al., 1994,FEBS Lett., 349(1): 23-8), 473-480, the entire disclosure of which isherein incorporated by reference).

For example, an isolated colony is inoculated in the liquid culturemedium such as the LB broth medium with the addition of antibiotics.After overnight incubation, the preculture can be used for seeding aculture of a larger volume. The expression of the polypeptide is theninduced, the cells develop over several hours and are then collected bycentrifugation. The cellular deposit can be lysed by chemical agentsknown in the art, or mechanically, e.g., by sonication. The protein canbe purified by means of its physicochemical properties as described forthe purification of recombinant FGF2 (Colin et al., 1997, Eur. J.Biochem., 249, 473-480, the entire disclosure of which is hereinincorporated by reference). If the protein is labeled with apolyhistidine tail, it can be purified via this tail by immobilizationon a chelating agent support of metallic ions, as described (Tang etal., Protein Expr. Purif. 1997 December; 11(3): 279-83, the entiredisclosure of which is herein incorporated by reference).

As a further example, expression vectors for expressing proteins havingpost-translational modifications such as glycosylation, such as theeukaryote systems (yeasts, plants, insects), can be used.

Thus, the recombinant protein can be produced, e.g., in the yeast Pichiapastoris as described by Sreekrishna et al. (1988, J. Basic Microbiol.,28(4): 265-78, the entire disclosure of which is herein incorporated byreference). The amplified DNA can be introduced in the same manner afterdigestion and ligation in an expression vector of Pichia pastoris,preferably containing a sequence coding for a selection marker. Asuitable Pichia pastoris vector is described in Scorer et al.,biotechnology (NY), 1994 February; 12(2): 181-4, the entire disclosureof which is herein incorporated by reference. The protein can eitherremain intracellular or can be secreted if the vector contains, at theend of the introduced gene, a sequence coding for a signal sequence ofsecretion such as, e.g., the prepropeptide factor of Saccharomycescerevisiae (Cregg et al., 1993; Scorer et al., 1993). A histidine tailcan also be added to one of the ends of the recombinant protein tofacilitate purification (Mozley et al., 1997, Photochem. Photobiol.665(5): 710-5).

The host is preferably selected from among: a bacterium, a yeast, aninsect cell, a mammal cell, or a plant cell.

The administration of therapeutic compositions comprising such proteinscan be implemented, e.g., via the topical, oral, intradermal,transdermal intra-ocular or intravenous route, or any other suitableenteral or parenteral route.

The fragments of the proteins can be used as antagonists of the proteinfrom which they originate. Thus, the administration to an animal of atherapeutic composition containing such fragments is recognized forinducing a diminution of the activity of the protein in the angiogenesismechanism for a given pathology.

We also provide a method for the diagnosis of an angiogenic pathology ina mammal, notably in a human being, consisting of detecting in the cellsof the mammal the overexpression or the underexpression of one or morenucleotide sequences identified by the numbers SEQ ID No. 1 to SEQ IDNo. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in theattached sequence listing.

Such a diagnostic method comprises the following steps:

-   -   detecting the expression of one or more of the nucleotide        sequences SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ        ID No. 284 to SEQ ID No. 290 in a cell population of a mammal;    -   detecting the expression of the same nucleotide sequences by a        reference cell population whose angiogenic state is known; and    -   identifying the differences in the level of expression in cells        of the same nucleotide sequences by the two cell populations.

We also provide a diagnostic and prognostic method for an angiogenicpathology in a mammal, notably in a human being, consisting of detectingin the cells of the mammal the overexpression or the underexpression ofone or more polypeptide sequences identified by the numbers SEQ ID No.54 to SEQ ID No. 102, or of the polypeptide sequences identified by thenumbers SEQ ID No. 291 to SEQ ID No. 297 in the attached sequencelisting.

As used herein, a “cell population of a mammal” is a collection ofmammalian cells of a certain type or lineage, or which are obtained fromthe same tissue or organ. It is understood that a cell population of amammal can comprise different cell types; for example, when thepopulation is obtained from the same tissue (e.g., blood) or organ(e.g., the liver). A cell population of a mammal can be obtained fromboth in vivo and in vitro (i.e., cultured cell) sources.

As used herein, a “reference cell population” is a collection of cellsof a certain type or lineage, or which are obtained from the same tissueor organ, for which the angiogenic state is known. It is understood thata “reference cell population” can comprise different cell types, and canbe obtained from both in vivo and in vitro sources.

As used herein, a gene is “overexpressed” when that gene produces anamount of RNA and/or corresponding protein in a cell population of amammal which is greater than the amount of RNA and/or correspondingprotein produced from the same gene in a reference cell population.

As used herein, a gene is “underexpressed” when that gene produces anamount of RNA and/or corresponding protein in a cell population of amammal which is less than the amount of RNA and/or corresponding proteinproduced from the same gene in a reference cell population.

According to a preferred aspect, the method comprises the followingsteps:

-   -   a) detecting one or more of polypeptide sequences SEQ ID No. 54        to SEQ ID No. 102, or polypeptide sequences identified by the        numbers SEQ ID No. 291 to SEQ ID No. 297, in a cell population        obtained from a mammal.    -   b) detecting the expression of these same polypeptide sequences        in a reference cell population whose angiogenic state is known;        and    -   c) identifying the differences in the level of expression of        these same polypeptide sequences in the two cell populations.

According to one particular aspect, in the diagnostic method, thedetection of the expression of the polypeptide sequences is performedafter the endothelial cells have been contacted with a biological fluidoriginating from a, patient.

We also provide a method for the verification of the therapeuticefficacy of an angiogenic treatment in a mammal, notably in a humanbeing, by the identification of a cell population in the mammal capableof overexpressing or underexpressing one or more nucleotide sequencesidentified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequence listing.

Such a method for the verification of therapeutic efficacy comprises thefollowing steps:

-   -   detecting the expression of one or more of nucleotide sequences        SEQ ID No. 1 to SEQ ID No. 53 and SEQ ID No. 225 in a cell        population from a mammal, to which is administered a therapeutic        composition intended to treat an angiogenic disorder;    -   detecting the expression of these same nucleotide sequences by a        reference cell population whose angiogenic state is known; and    -   identifying the differences in the level of expression of these        same nucleotide sequences in the two cell populations.

According to preferred modes of implementation, the verification methodis performed on a cell population from a mammal in vivo, ex-vivo or on acell population isolated from the mammal in vitro.

According to one particular aspect, in the verification method, thedetection of the expression of the sequences is performed after havingcontacted the endothelial cells with a biological fluid obtained from apatient.

This disclosure also pertains to a method for screening for compoundsuseful for treating an angiogenic pathology of a mammal, notably a humanbeing.

According to one preferred mode of implementation, such a screeningmethod comprises the following steps:

-   -   detecting the expression of one more of nucleotide sequences SEQ        ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to        SEQ ID No. 290 in a cell population contacted with a compound        capable of having a therapeutic effect on an angiogenic        pathology;    -   detecting the expression of these same nucleotide sequences in a        reference cell population whose angiogenic state is known; and    -   identifying the differences in the level of expression of these        same nucleotide sequences in the two cell populations.

According to another preferred aspect, such a screening method alsocomprises the following steps:

-   -   detecting the expression of one or more of polypeptide sequences        identified by the numbers SEQ ID No. 54 to SEQ ID No. 102, or        with the polypeptide sequences identified by the numbers SEQ ID        No. 291 to SEQ ID No. 297, in the attached sequence listing by a        cell population contacted with a compound capable of having, or        which has, a therapeutic effect on an angiogenic pathology;    -   detecting the expression of these same polypeptide sequences in        a reference cell population whose angiogenic state is known; and    -   identifying the differences in the level of expression of these        same polypeptide sequences in the two cell populations.

As used herein, a compound has a “therapeutic effect” on an angiogenicpathology when, upon administration of that compound to an individualsuffering from an angiogenic pathology, the symptoms of the angiogenicpathology are lessened, prevented or otherwise alleviated, or the growthof new blood vessels in the region of the angiogenic pathology is slowedor halted. In the practice of the present method, it is understood thata test compound which causes a difference in the expression ofnucleotide sequences between a cell population of a mammal and areference population indicates that the test compound has a therapeuticeffect on an angiogenic pathology.

According to one particular aspect, in the screening method thedetection of expression of the sequences is performed after contactingthe endothelial cells with a biological fluid obtained from a patient.

The following can be cited among the angiogenic pathologies (also called“angiogenic disorders”) that could be diagnosed or treated with themethods and pharmaceutical compositions: tumor vascularization,retinopathies (e.g., diabetic retinopathy), rheumatoid arthritis,Crohn's disease, atherosclerosis, hyperstimulation of the ovary,psoriasis, endometriosis associated with neovascularization, restenosisdue to balloon angioplasty, tissue overproduction due to cicatrization,peripheral vascular disease, hypertension, vascular inflammation,Raynaud's disease and phenomena, aneurism, arterial restenosis,thrombophlebitis, lymphangitis, lymphedema, cicatrization and tissuerepair, ischemia, angina, myocardial infarction, chronic heart disease,cardiac insufficiencies such as congestive heart failure, age-linkedmacular degeneration and osteoporosis.

We also provide a device comprising a support. The support comprises oneor more probes specific of one or more nucleotide sequences identifiedby the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQ ID No. 225 and SEQ IDNo. 284 to SEQ ID No. 290 in the attached sequence listing, or fragmentsor derivatives thereof, for the implementation of the screening method.

In the context of this disclosure, the term “probe” is understood tomean any single-strand DNA fragment the sequence of which iscomplementary to a target sequence: this target sequence thus can bedetected by hybridization with the labeled probe (labeled byincorporation of, e.g., radioactive atoms or fluorescent groups), whichplay the role of a molecular “fish hook”.

According to preferred aspects, the support of a device is selected fromamong a glass membrane, a metal membrane, a polymer membrane or a silicamembrane.

Devices can be, e.g., DNA chips comprising one or more nucleotidesequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequencelisting.

We also provide a kit intended for measuring the differential display ofgenes implicated in angiogenic pathologies, comprising a device asdescribed above, specific primers and the accessories required for theamplification of the sequences extracted from a sample, hybridizationwith the probes of the device and the performance of the differentialdisplay measurements.

We also provide a kit intended for the measurement of the differentialdisplay of genes implicated in angiogenic disorders, comprising as areference a cell population genetically modified cell line expressing,in a stable manner, a vector comprising at least one of the nucleotidesequences identified by the numbers SEQ ID No. 1 to SEQ ID No. 53, SEQID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in the attached sequencelisting, or one of their fragments or derivatives, and the meansrequired for measuring the differential display.

We also provide a kit intended for the measurement of the differentialdisplay of genes implicated in angiogenic disorders, comprising as areference cell population a genetically modified cell line expressing ina stable manner the vector expressing at least one antisense sequence ofthe nucleotide sequences identified by the numbers SEQ ID No. 1 to SEQID No. 53, SEQ ID No. 225 and SEQ ID No. 284 to SEQ ID No. 290 in theattached sequence listing, or one of their fragments or derivatives, andthe means required for the measurement of the differential display.

Verification that the fifty-four genes identified are implicated in themechanism of angiogenesis was performed according to the methodologydescribed in the Material and Methods section below, and is illustratedby means of attached FIGS. 1 to 11 in which:

-   -   FIG. 1 shows that the expression of GS-V1, GS-V2, GS-V4, GS-V5        and GS-V15 in human endothelial cells inhibits the formation of        capillary tubes. This Figure shows endothelial cells transfected        with: 1A) GS-V1 coding for the specific antisense transcript of        GS-N1; 1B) GS-V2 coding for the specific antisense transcript of        GS-N2; 1C) GS-V4 coding for the specific antisense transcript of        GS-N4; 1D) GS-V5 coding for the specific antisense transcript of        GS-N5; 1E) GS-V15 coding for the specific antisense transcript        of GS-N15; and 1F) the empty vector (control).    -   FIG. 2 shows that the expression of GS-V3 and GS-V14 in human        endothelial cells inhibits the formation of capillary tubes.        This Figure shows endothelial cells transfected with: 2A) GS-V3        coding for the specific antisense transcript of GS-N3; 2B)        GS-V13 coding for the specific antisense transcript of GS-N13;        and 2C) the empty vector (control).    -   FIG. 3 shows that the expression of GS-V6, GS-V8 and GS-V10 in        human endothelial cells induces the formation of capillary        tubes. This Figure shows transfected endothelial cells in which:        3A) GS-V6 coding for the specific antisense transcript of GS-N6;        3B) GS-V8 coding for the specific antisense transcript of GS-N8;        3C) GS-V10 coding for the specific antisense transcript of        GS-N10 and its homologue GS-N54; and 3D) the empty vector        (control).    -   FIG. 4 shows that the expression of GS-V7, GS-V9, GS-V1, GS-V12        and GS-V14 in human endothelial cells inhibits the formation of        capillary tubes. This Figure shows endothelial cells transfected        with: 4A) GS-V7 coding for the specific antisense transcript of        GS-N7; 4B) GS-V9 coding for the specific antisense transcript of        GS-N9; 4C) GS-V11 coding for the specific antisense transcript        of GS-N11; 4D) GS-V12 coding for the specific antisense        transcript of GS-N12; and 4E) GS-V14 coding for the specific        antisense transcript of GS-N14 and 4F) the empty vector        (control).

FIG. 5 shows that the expression of GS-V16, GS-V17, GS-V18, GS-V19 andGS-V21 in human endothelial cells inhibits the formation of capillarytubes. This Figure shows endothelial cells transfected with: 5A) GS-V16coding for the specific antisense transcript of GS-N16; 5B) GS-V17coding for the specific antisense transcript of GS-N17; 5C) GS-V18coding for the specific antisense transcript of GS-N18; 5D) GS-V19coding for the specific antisense transcript of GS-N19; 5E) GS-V21coding for the specific antisense transcript of GS-N21; and 5F) theempty vector (control).

-   -   FIG. 6 shows that the expression of GS-V22, GS-V24, GS-V25,        GS-V26 and GS-V27 in human endothelial cells inhibits the        formation of capillary tubes. This Figure shows endothelial        cells transfected with: 6A) GS-V22 coding for the specific        antisense transcript of GS-N22; 6B) GS-V24 coding for the        specific antisense transcript of GS-N24 and of its homologue        GS-N49; 6C) GS-V25 coding for the specific antisense transcript        of GS-N25 and of its homologue GS-N50; 6D) GS-V26 coding for the        specific antisense transcript of GS-N26; 6E) GS-V27 coding for        the specific antisense transcript of GS-N27 and of its homologue        GS-N51; and 6F) the empty vector (control).    -   FIG. 7 shows that the expression of GS-V28, GS-V29, GS-V30,        GS-V31 and GS-V32 in human endothelial cells inhibits the        formation of capillary tubes. This Figure shows endothelial        cells transfected with: 7A) GS-V28 coding for the specific        antisense transcript of GS-N28; 7B) GS-V29 coding for the        specific antisense transcript of GS-N29 and of its homologue        GS-N52; 7C) GS-V30 coding for the specific antisense transcript        of GS-N30; 7D) GS-V31 coding for the specific antisense        transcript of GS-N31 and of its homologue GS-N53; 7E) GS-V32        coding for the specific antisense transcript of GS-N32; and 7F)        the empty vector (control).    -   FIG. 8 shows that the expression of GS-V33, GS-V34, GS-V35,        GS-V37 and GS-V38 in human endothelial cells inhibits the        formation of capillary tubes. This Figure shows endothelial        cells transfected with: 8A) GS-V33 coding for the specific        antisense transcript of GS-N33; 8B) GS-V34 coding for the        specific antisense transcript of GS-N34; 8C) GS-V35 coding for        the specific antisense transcript of GS-N35; 8D) GS-V37 coding        for the specific antisense transcript of GS-N37; 8E) GS-V38        coding for the specific antisense transcript of GS-N38; and 8F)        the empty vector (control).    -   FIG. 9 shows that the expression of GS-V40, GS-V42, GS-V43,        GS-44 and GS-V45 in human endothelial cells inhibits the        formation of capillary tubes. This Figure shows endothelial        cells transfected with: 9A) GS-V40 coding for the specific        antisense transcript of GS-N40; 9B) GS-V42 coding for the        specific antisense transcript of GS-N42; 9C) GS-V43 coding for        the specific antisense transcript of GS-N43; 9D) GS-V44 coding        for the specific antisense transcript of GS-N44; 9E GS-V45        coding for the specific antisense transcript of GS-N45; and 9F)        the empty vector (control).    -   FIG. 10 shows that the expression of GS-V20, GS-V23, GS-V36,        GS-V39 and GS-V41 in human endothelial cells inhibits the        formation of capillary tubes. This Figure shows endothelial        cells transfected with: 10A) GS-V20 coding for the specific        antisense transcript of GS-N20; 10B) GS-V23 coding for the        specific antisense transcript of GS-N23 and of its homologues        GS-N47 and GS-N48; 10C) GS-V36 coding for the specific antisense        transcript of GS-N36; 10D) GS-V39 coding for the specific        antisense transcript of GS-N39; 10E) GS-V41 coding for the        specific antisense transcript of GS-N41; and 10F) the empty        vector.    -   FIG. 11 shows that the expression of GS-V46 in human endothelial        cells inhibits the formation of capillary tubes. This Figure        shows endothelial cells transfected with: 11A) GS-V46 coding for        the specific antisense transcript of GS-N46; and 11B) the empty        vector (control).

This disclosure will now be illustrated by the following non-limitingexamples.

EXAMPLES Material and Methods

1. Culture of the Cells and Angiogenesis Test

Human endothelial cells from umbilical veins (HUVEC) grown under thefollowing four culture conditions were used for identifying the genescoding for the cellular constituents implicated in the regulation ofangiogenesis:

-   -   A control condition in which the endothelial cells are not        stimulated.    -   An angiogenic condition in which the endothelial cells are        stimulated by one or more angiogenic factors.    -   An angiogenesis inhibition condition in which the endothelial        cells are stimulated by one or more angiogenic factors and        brought into the presence of one or more angiostatic conditions.    -   Another control condition in which the endothelial cells are        stimulated by one or more angiostatic factors.

The endothelial cells were maintained in complete medium (EGM-2 fromClonetics).

For the identification of the genes implicated in angiogenesis, the invitro test of angiogenesis according to the model of Montesano et al.(1986, Proc. Natl. Acad. Sc. USA, 83(19): 7297-301, the entiredisclosure of which is herein incorporated by reference) was used.Briefly, the cells were first sown on a gel type I collagen in completemedium until confluence. The reference HUVEC cells were then cultured onserum-impoverished medium without growth factors: EBM-2+2% serum anddifferent factors were added under the test conditions, as follows:

-   -   FEG2: at concentrations between about 5 ng/ml and about 60        ng/ml, preferably between about 10 and about 40 ng/ml; VEGF: at        concentrations between about 10 ng/ml and about 60 ng/ml,        preferably between about 30 ng/ml and about 50 ng/ml; PF4: at        concentrations between about 0.1 and about 5 μg/ml, preferably        between about 0.5 μg/ml and about 1 μg/ml; TNF-α at        concentrations between about 20 ng/ml and about 100 ng/ml,        preferably between about 30 ng/ml and about 60 ng/ml; IFN-γ: at        concentrations between about 50 ng/ml and about 200 ng/ml,        preferably between about 80 ng/ml and about 120 ng/ml.

The human endothelial cells placed under the four previously mentionedculture conditions were then used for identifying genes coding for thecellular constituents implicated in the regulation of angiogenesis.

2. Angiogenic and Angiostatic Factors

Angiogenic and angiostatic factors having an effect on the expression ofthe genes identified in correlation with the formation of neovessels orthe inhibition of neovessels, respectively, used as an example in theframework are illustrated below:

-   -   VEGF=vascular endothelial growth factor.    -   FGF2=basic fibroblast growth factor.    -   HGF=hepatocyte growth factor.    -   PF4=platelet factor 4.    -   IFN-γ=interferon gamma.    -   TNF-α=tumor necrosis factor alpha.

TNF-α is a regulator of angiogenesis. It can induce angiogenesis in vivobut also inhibit the formation of vessels in vitro (Frater-Schroder etal., 1987, Proc. Natl. Acad. Sci. USA, 84(15): 5277-81; Fajardo et al.,1992, Am. J. Pathol. March, 140 (3): 539-44; Niida et al., 1995, Neurol.Med. Chir. (Tokyo), 35(4): 209-14). In our in vitro model ofangiogenesis, TNF-α is used under angiogenesis inhibition conditions.

3. Comparison of the Gene Expressions

Gene expression can be compared, for example, using the DNA chips, SAGE,an amplification reaction by quantitative PCR, viral vectors forconstructing subtractive banks or analysis by differential display.

In the context of the experimental studies presented below, thedifferential display technique for the identification of the genes waspreferentially used.

Differential Display

Total RNAs were prepared from HUVEC cells cultured on a collagen gel inthe presence of the different factors used, according to the RNeasy Minikit (Qiagen) integrating a step of DNase I digestion according to theprotocol recommended by the manufacturer.

Differential display from total RNAs was performed according to themethod described by Liang and Pardee (1992, Science, 14: 257(5072):967-7) using aP33-ATP in isotopic dilution during the PCR amplificationfor the visualization of the bands by autoradiography of theelectrophoresis gels.

Thus the DNA fragments differentially present on the gel as a functionof the culture conditions were cut, reamplified, cloned in plasmid PGEMeasy vector (Promega), sequenced and identified by querying the BLASTdatabase.

4. Verification of the Implication of the Genes Identified in theMechanism of Angiogenesis Functionality Test of the Genes

In a second step, the functionality of each gene sequence identified wastested in the in vitro angiogenesis model discussed above withendothelial cells transfected with an expression vector comprising anantisense oligonucleotide of the sequence.

For the construction of these vectors, specific primers for each of theidentified sequences were designed. These primers are indicated in TableI below, and are identified with the sequence numbers SEQ ID No. 195 toSEQ ID No. 222, SEQ ID No. 226 to SEQ ID No. 283 and SEQ ID No. 298 toSEQ ID No. 299 in the attached sequence listing.

TABLE I SEQUENCE ID Primer name SEQ ID No 1 (GS-N1) GV1-1 GV1-2 SEQ IDNo 2 (GS-N2) GV2-1 GV2-2 SEQ ID No 3 (GS-N3) GV3-1 GV3-2 SEQ ID No 4(GS-N4) GV4-1 GV4-2 SEQ ID No 5 (GS-N5) GV5-1 GV5-2 SEQ ID No 6 (GS-N6)GV6-1 GV6-2 SEQ ID No 7 (GS-N7) GV7-1 GV7-2 SEQ ID No 8 (GS-N8) GV8-1GV8-2 SEQ ID No 9 (GS-N9) GV9-1 GV9-2 SEQ ID No 10 (GS-N10) GV10-1GV10-2 SEQ ID No 11 (GS-N11) GV11-1 GV11-2 SEQ ID No 12 (GS-N12) GV12-1GV12-2 SEQ ID No 13 (GS-N13) GV13-1 GV13-2 SEQ ID No 14 (GS-N14) GV14-1GV14-2 SEQ ID No 15 (GS-N15) GS-PGS-F GS-PGM-R SEQ ID No 16 (GS-N54)GV10-1 GV10-2 SEQ ID No 17 (GS-N16) GV16-1 GV16-2 SEQ ID No 18 (GS-N17)GV17-1 GV17-2 SEQ ID No 19 (GS-N18) GV18-1 GV18-2 SEQ ID No 20 (GS-N19)GV19-1 GV19-2 SEQ ID No 21 (GS-N20) GV20-1 GV20-2 SEQ ID No 22 (GS-N21)GV21-1 GV21-2 SEQ ID No 23 (GS-N22) GV22-1 GV22-2 SEQ ID No 24 (GS-N23)GV23-1 GV23-2 SEQ ID No 25 (GS-N24) GV24-1 GV24-2 SEQ ID No 26 (GS-N25)GV25-1 GV25-2 SEQ ID No 27 (GS-N26) GV26-1 GV26-2 SEQ ID No 28 (GS-N27)GV27-1 GV27-2 SEQ ID No 29 (GS-N28) GV28-1 GV28-2 SEQ ID No 30 (GS-N29)GV29-1 GV29-2 SEQ ID No 31 (GS-N30) GV30-1 GV30-2 SEQ ID No 32 (GS-N31)GV31-1 GV31-2 SEQ ID No 33 (GS-N32) GV32-1 GV32-2 SEQ ID No 34 (GS-N33)GV33-1 GV33-2 SEQ ID No 35 (GS-N34) GV34-1 GV34-2 SEQ ID No 36 GS-N35)GV35-1 GV35-2 SEQ ID No 37 (GS-N36) GV36-1 GV36-2 SEQ ID No 38 (GS-N37)GV37-1 GV37-2 SEQ ID No 39 (GS-N38) GV38-1 GV38-2 SEQ ID N° 40 (GS-N39)GV39-1 GV39-2 SEQ ID No 41 (GS-N40) GV40-1 GV40-2 SEQ ID No 42 (GS-N41)GV41-1 GV41-2 SEQ ID No 43 (GS-N42) GV42-1 GV42-2 SEQ ID No 44 (GS-N43)GV43-1 GV43-2 SEQ ID No 45 (GS-N44) GV44-1 GV44-2 SEQ ID No 46 (GS-N45)GV45-1 GV45-2 SEQ ID No 47 (GS-N46) GS-PGM-F GS-PGS-R SEQ ID No 48(GS-N47) GV23-1 GV23-2 SEQ ID No 49 (GS-N48) GV23-1 GV23-2 SEQ ID No 50(GS-N49) GV24-1 GV24-2 SEQ ID No 51 (GS-N51) GV27-1 GV27-2 SEQ ID No 52(GS-N52) GV29-1 GV29-2 SEQ ID No 53 (GS-N53) GV31-1 GV31-2 SEQ ID No 225(GS-N50) GV50-1 GV50-2

These primers contain, at each of their ends, a different restrictionenzyme site (SalI: GTCGAC or MluI: ACGCGT).

Amplified fragments of each gene were obtained by PCR from each of thebacterial plasmids containing the fragment of the gene identified usingthe primers.

These fragments were purified, digested by the restriction enzymes SalIand MluI and inserted in a mammalian expression vector of the typepCi-neo vector (Promega), which is itself digested by one of these tworestriction enzymes.

Each fragment was introduced in the antisense orientation.

In the particular cases of the GS-N15 and GSS-N46 sequences, theamplification of the fragment cloned in the bacterial plasmid wasperformed by means of particular primers selected from among thesequences GS-PGS-F, GS-PGM-RX or GS-PGM-F and GS-PGS-R, hybridizing atthe regions of the plasmid surrounding the cloned gene and also havingin their ends restriction sites (SalI and MluI) not contained in thecloned fragment or present in the multisite region of the expressionvector.

These two restriction sites could be interchanged, depending on whetherthe fragment was cloned in the bacterial plasmid in its sense orantisense orientation.

Controls performed with these primers, which can be considered universalprimers, in the absence of the cloned gene (empty plasmid) showed thatthe amplified fragment of the plasmid (40 bp), when it is integrated inthe expression vector, does not alter the formation of the neovessels inthe in vitro functionality test. The results obtained with vectorsconstructed in this manner were identical to those obtained with theempty vector and show that these supplementary base pairs do not alterthe effect of the specific antisense fragments of the identifiedsequence.

Generally speaking, vectors that can be used for demonstrating thefunctionality of the identified genes in the mechanisms of angiogenesiscomprise any mammalian expression vector system. Suitable expressionvectors can also comprise a promoter that enables expression of a clonedgene; for example, the strong promoter of human cytomegalovirus (CMV).

Other constitutive or inducible expression vectors that can be used arespecified in the nonexhaustive list below:

Vectors marketed by the company Promega; vectors with a strong promoterfor a high level of expression constitutive of genes in mammal cells(pCI Mammalian Expression vector, Expression Vector System cloningvector pALTER®*-MAX); vectors marketed by the company Invitrogen:(pcDNA3.1, -/hygro, -/Zeo, pcDNA4/HisMAx, -E, pBudCE4, pRcRSV, pRcCMV2,pSecTag2, -/hygro secretion vectors, the vectors pEBVHis A, B and C);expression vectors for mammals marketed by the company Clontech (PIRES,pIRES-EYFP pIRES2-EGFP, pCMV-Myc and pCMV-HA); Epitope-tagged pTRE; thevectors VP16 Minimal Domain (ptTA 2, ptTA 3 and ptTA 4); thebidirectional Tet expression vectors (pBI, pBI-EGFP, pBI-G, pBI-L),pRevTRE, pTRE2, pLEGFP-N1 Retroviral Vector pLEGFP-C1; and theadenovirus expression systems Adeno-X, pCMS-EGFP, pd1EGFP-N1,pd2ECFP-N1, pd2EYFP-N1, pEGFP (-C1, -C2, -C3, -N1, -N2, -N3), pEYFP-C1,-N1.

Each vector comprising the antisense fragment was then replicated in E.coli, extracted, purified and quantified. One μg of each vector wasincubated in the presence of a transfectant agent (Effectene, Qiagen)following the protocol recommended by the manufacturer for endothelialcells. Twenty-four hours after the transfection, the endothelial cellswere trypsinized and spread on the extracellular matrix containing theangiogenic factors with Matrigel according to the model described byGrant et al. (1989, Cell, 58(5): 933-43, the entire disclosure of whichis herein incorporated by reference). After 24 h of incubation, theformation of vessels was observed and compared to the control cellstransfected with the empty mammalian expression vector.

5. Establishment of a Bank of Stable Lines Expressing the VectorsContaining the Gene Sequences or Their Fragments or Their AntisenseSequences.

The expression systems can comprise an antibiotic selection markercomprising an antibiotic resistance gene, for selecting the transfectedcells, stably expressing the vector comprising the nucleic acid clonedin the vector—either in the same vector or in a second co-transfectedvector.

This expression vector can be a constitutive or inducible expressionsystem.

In the particular example described below, stable cell lines for theexpression of the antisense oligonucleotide corresponding to eachidentified gene were obtained with a constitutive expression vector,after selection in presence of antibiotic.

To implement this selection, 24 h after the transfection performed underthe conditions described above, the BAEC endothelial cells weretrypsinized and sown at the rate of 80,000 cells/well in six-well platesin the presence of 700 μg/ml of the antibiotic G418 (Promega). A controlwell was sown with the untransfected cells. The medium was changed everythree days with a recharge of the antibiotic. The control cells wereeliminated after 8 to 10 days; the antibiotic-resistant cells werecollected at confluence (after 2 to 3 weeks) then transferred intoculture flasks, still in the presence of the antibiotic. The stable celllines were then tested for their capacity to form or not form vessels inthe in vitro angiogenesis test discussed above.

6. Results

6.1 Identification of the Genes

The nucleic acid sequences designated GS-N1, GS-N2, GS-N3, GS-N4, GS-N5,GS-N6, GS-N7, GS-N8, GS-N9, GS-N10 (or GS-N54), GS-N11, GS-N12, GS-N13,GN-14 and GS-N15, and respectively the proteins coded by the nucleicacids GS-N1 to GS-N13 and GS-N15 designated: angioinducine,angiodockine, angioblastine, angioreceptine, angiodensine,angiopartnerine, vassoserpentine, angiosulfatine, vassoreceptine,angiokinasine, vassosubstratine, angiosignaline, angiofoculine,angiohelicine and angioacyline, have not previously been identified ashaving any biological role, let alone in the process of angiogenesis ordifferentiation of endothelial cells into capillary tubes. These nucleicacids and proteins are described below.

The previously described differential display method enabledidentification of the following mRNAs.

-   -   GS-N1: a 1683-bp mRNA identified by the sequence SEQ ID No. 1 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number BC008502.

The sequence of this mRNA has a coding sequence from the nucleotide 159to the nucleotide 458. A protein GS-P1 resulting from the translation ofthis mRNA was thus identified. This protein is composed of 99 aminoacids (aa), identified by the number SEQ ID No. 54 in the attachedsequence listing and designated Angioinducine.

-   -   GS-N2: a 1649-bp mRNA identified by the sequence SEQ ID No. 2 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number        NM_(—)022823.

The sequence of this mRNA has a coding sequence from nucleotide 367 tonucleotide 1071. A protein GS-P2 resulting from the translation of thismRNA was thus identified. This protein is composed of 234 aa, identifiedby the number SEQ ID No. 55 in the attached sequence listing anddesignated Angiodockine.

-   -   GS-N3: a 5766-bp nmRNA identified by the sequence SEQ ID No. 3        in the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number AB007963.

The sequence of this mRNA has a coding sequence from nucleotide 978 tonucleotide 2465. A protein GS-P3 resulting from the translation of thismRNA was thus identified. This protein is composed of 495 aa, identifiedby the number SEQ ID No. 56 in the attached sequence listing anddesignated Angioblastine.

-   -   GS-N4: a 5242-bp mRNA identified by the sequence SEQ ID No. 4 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it by the accession number        AB037835.

The sequence of this mRNA has a partial coding sequence from nucleotide1 to nucleotide 4762. A protein GS-P4 resulting from the translation ofthis mRNA was thus identified. This protein is composed of 1586 aa,identified by the number SEQ ID No. 57 in the attached sequence listingand designated Angioreceptine.

-   -   GS-N5: a 2153-bp mRNA identified by the sequence SEQ ID No. 5 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it by the accession number        AK025682.

The sequence of this mRNA has a coding sequence from nucleotide 39 tonucleotide 691. A protein GS-P5 resulting from the translation of thismRNA was thus identified. This protein is composed of 217 aa, identifiedby the number SEQ ID No. 58 in the attached sequence list and designatedAngiodensine.

-   -   GS-N6: a 3005-bp mRNA identified by the sequence SEQ ID No. 6 in        the attached sequence listing. A BLAST search on the GENBANK        database identified it as accession number AK023284.

The sequence of this mRNA has a coding sequence from nucleotide 90 tonucleotide 773. A protein GS-P6 resulting from the translation of thismRNA was thus identified. This protein is composed of 227 aa, identifiedby the number SEQ ID No. 59 in the attached sequence listing anddesignated Vassoserpentine.

-   -   GS-N7: a 4397-bp mRNA identified by the sequence SEQ ID No. 7 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number AB033073.

The sequence of this mRNA has a partial coding sequence from nucleotide286 to nucleotide 2943. A protein GS-P7 resulting from the translationof this mRNA was thus identified. This protein is composed of 885 aa,identified by the number SEQ ID No. 60 in the attached sequences listingand designated Angiosulfatine.

-   -   GS-N8: a 5844-bp mRNA identified by the sequence SEQ ID No. 8 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number AB023187.

The sequence of this mRNA has a partial coding sequence from nucleotide1 to nucleotide 3456. A protein GS-P8 resulting from the translation ofthis mRNA was thus identified. This protein is composed of 1151 aa,identified by the number SEQ ID No. 61 in the attached sequence listingand designated Vassoreceptine.

-   -   GS-N9: a 4266-bp mRNA identified by the sequence SEQ ID No. 9 in        the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number AB014587.

The sequence of this mRNA has a partial coding sequence from nucleotide1 to nucleotide 3528. A protein GS-P9 resulting from the translation ofthis mRNA was thus identified. This protein is composed of 1175 aa,identified by the number SEQ. ID No. 62 in the attached sequence listingand designated Angiokinasine.

Angiokinasine is homologous with MAP4K4 (SEQ ID No. 224 in the attachedsequence listing), accession number: XM_(—)038751 (nucleic sequence:4197 bp, protein sequence: 1141 aa). Thus, we provide a heretoforeunknown role for MAP4K4 in the regulation f angiogenesis.

-   -   GS-N10: a 2034-bp mRNA identified by the sequence SEQ ID No. 10        in the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number        XM_(—)035658.

This sequence is homologous with the sequence GS-N54.

-   -   GS-N54: a 4749-bp mRNA identified by the sequence SEQ ID No. 16        in the attached sequence listing. A BLAST search on the GENBANK        sequence database identified as accession number AK024248.

The sequence of mRNA GS-N10 has a coding sequence from nucleotide 618 tonucleotide 1787. A protein GS-P10 resulting from the translation of thismRNA was thus identified. This protein is composed of 389 aa, identifiedby the number SEQ ID No. 63 in the attached sequence listing anddesignated Vassosubstratine.

-   -   GS-N11: a 1817-bp mRNA identified by the sequence SEQ ID No. 11        in the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number        NM_(—)032181.

The sequence of this mRNA has a coding sequence from nucleotide 439 tonucleotide 897. A protein GS-P11 resulting from the translation of thismRNA was thus identified. This protein is composed of 152 aa, identifiedunder the number SEQ ID No. 64 in the attached sequence listing anddesignated Angiosignaline.

-   -   GS-N12: a 4131-bp mRNA identified by the sequence SEQ ID No. 12        in the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number AB023233.

The sequence of this mRNA has a partial coding sequence from nucleotide1 to nucleotide 2834. A protein GS-P12 resulting from the translation ofthis mRNA was thus identified. This protein is composed of 793 aa,identified under the number SEQ ID No. 65 in the attached sequencelisting and designated Angiofoculine.

-   -   GS-N13: a 2566-bp mRNA identified by the sequence SEQ ID No. 13        in the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as number XM_(—)018273.

The sequence of this mRNA has a coding sequence from nucleotide 426 tonucleotide 2345. A protein GS-P13 resulting from the translation of thismRNA was thus identified. This protein is composed of 639 aa, identifiedby the number SEQ ID No. 66 in the attached sequence listing anddesignated Angiohelicine.

-   -   GS-N14: a 1830-bp mRNA identified by the sequence SEQ ID No. 14        in the attached sequence listing. A BLAST search on the GENBANK        sequence data base identified it as accession number AK022109.

This sequence does not contain a coding sequence.

-   -   GS-N15: a 6253-bp mRNA identified by the sequence SEQ ID No. 15        in the attached sequence listing. A BLAST search on the GENBANK        sequence database identified it as accession number        NM_(—)014873.

The sequence of this mRNA has a coding sequence from nucleotide 228 tonucleotide 1340. A protein GS-P15 resulting from the translation of thismRNA was thus identified. This protein is composed of 370 aa, identifiedunder the number SEQ ID No. 67 in the attached sequence listing anddesignated Angioacyline.

The nucleic acid sequences designated GS-N16 to GS-N53 identified by thenumbers SEQ ID No. 17 to SEQ ID No. 53 and SEQ ID No. 225 in theattached sequence listing, and respectively the proteins coded by thenucleic acids, identified by the numbers SEQ ID No. 68 to SEQ ID No. 102in the attached sequence listing, had not previously been identified ashaving a biological role in the process of angiogenesis or thedifferentiation of endothelial cells into capillary tubes. Thesesequences are described below.

-   -   GS-N16: a 3139-bp mRNA identified by the sequence number SEQ ID        No. 17 in the attached sequence listing. A BLAST search of the        GENBANK sequence database identified it as accession number        XM_(—)011833 (Homo sapiens phosducin-like (PDCL)).

The sequence of this mRNA has a coding region from nucleotide 167 tonucleotide 1072. A protein GS-P16 resulting from the translation of thismRNA was thus identified.

This protein whose sequence is identified by the number SEQ ID No. 68 inthe attached sequence listing is an analogue of the phosducin designatedPDCL composed of 301 aa.

-   -   GS-N17: a 2326-bp mRNA identified as sequence number SEQ ID No.        18 in the attached sequence listing. A BLAST search of the        GENBANK sequence database identified it as access number        BC011860 (Ribosome Protein L3 (RPL3)).

The sequence of this mRNA has a coding sequence from nucleotide 1030 to2241 identified by number SEQ ID No. 69 in the attached sequencelisting. The polypeptide sequence of 403 aa is identical (100% identity)to the human ribosome protein L3 (RPL3) whose nucleic sequence isshorter, accession number BC006483 (SEQ ID No. 285), BC012786 (SEQ IDNo. 286).

The ribosome protein L3 (RPL3) is a highly conserved protein (Herwig etal., 1992, Eur. J. Biochem. 207(3): 877-85; Van Raay et al., 1996,Genomics: 37(2): 172-6) localized in the large ribosome subunit. In E.coli this protein is known to bind ribosome RNA 23S and participate inthe formation of the peptidyltransferase center of this ribosome(Noller, 1993, Bacteriol. 175: 5297-53039; Noller, 1997, Ann. Rev.Biochem. 66: 679-716).

Although the differential expression of the ribosome protein L3 has beendemonstrated in many studies: in skeletal muscle in an obesity studymodel in the mouse (Vicent et al., 1998, Diabetes 47: 1451-8), in thehypothalamus and brown adipose tissues in the mouse implicating RLP3 inthe regulation of energy equilibrium (Allan et al., 2000, PhysiologicalGenomics, 3: 149-156), there have been no descriptions of differentialexpression during angiogenesis nor in the regulation of angiogenesis.

-   -   GS-N18: a 3937-bp mRNA identified as sequence number SEQ ID No.        19 in the attached sequence listing. A BLAST search of the        GENBANK sequence database identified it as accession number        XM_(—)042798 (Protein 20 RING FINGER protein 20 (RNF20)).

This mRNA presents a coding sequence from nucleotide 89 to nucleotide3016. Its direct translation enables identification of a proteinhomologous with the protein RNF20 composed of 975 aa (GS-P18),identified by the number SEQ ID No. 70 in the attached sequence listing.

This sequence GS-P18 presents a total identity with the protein sequencededuced from the nucleotide sequence identified by accession numberAF265230 in the GENBANK database and by the number SEQ ID No. 287 in theattached sequence listing. The nucleotide sequences corresponding to thetwo proteins present a homology of 99% with each other.

The protein RNF20 is still poorly understood but it is characterized bythe presence of the RING FINGER domain. The ring finger proteins couldplay a role in the formation and architecture of large protein complexesthat contribute to various cellular processes, such as transduction ofthe signal, oncogenesis, apoptosis, development, differentiation,regulation of genes, ubiquination (Saurin et al., 1996, Trends Biochem.Sci. 21, 208-214; Borden, 2000, J. Mol. Biol., 295, 1103-1112; Topcu etal., 1999, Oncogene 18, 7091-7100).

It has not been described to date that the protein RNF20 is implicatedin the regulation of angiogenesis.

-   -   GS-N19: a 2167-bp mRNA identified by number SEQ ID No. 20 in the        attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number BC002781        (protein analogue of the splice factor, arginine/serine-rich 4).

The sequence of this mRNA presents a coding region from nucleotide 107to nucleotide 1591. A protein GS-P19 resulting from the translation ofthis mRNA, presented as the number SEQ ID No. 71 in the attachedsequence listing, was thus identified. This protein, composed of 494 aa,is homologous with the splice factor arginine/serine-rich 4 (SFRS4)(which is also called SRp75) and presents the same characteristicdomains.

-   -   GS-N20: a 5801-bp mRNA identified as number SEQ ID No. 21 in the        attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number U65090        (carboxypeptidase D).

The sequence of this mRNA has a coding region from nucleotide 36 tonucleotide 4169. A protein GS-P21, stemming from the direct translationof this mRNA, presented as number SEQ ID No. 72 in the attached sequencelisting, was thus identified. This protein, called carboxypeptidase D,is composed of 1377 aa.

-   -   GS-N21: a 8171-bp mRNA identified as number SEQ ID No. 22 in the        attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number NM_(—)004652        (protease 9 specific of ubiquitin).

The sequence of this mRNA has a coding sequence from nucleotide 60 tonucleotide 7751. There was thus identified a protein resulting from thetranslation of this mRNA. This protein, designated GS-P21, protease 9specific of ubiquitin, is composted of 2563 aa. It is identified asnumber SEQ ID No. 73 in the attached sequence listing.

-   -   GS-N22: a 3851-bp mRNA identified as number SEQ ID No. 23 in the        attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number NM_(—)002525        (nardilysine (N-arginine dibasic convertase) (NRD1)).    -   GS-N23: a 13,107-bp mRNA identified by the number SEQ ID No. 24        in the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number XM_(—)016303        (mRNA of myeloid/lymphoid or mixed lineage leukemia (MLL)).

The mRNA sequence identified by the number SEQ ID No. 24 has a codingsequence from nucleotide 1872 to nucleotide 10,001. A protein GS-P23resulting from the translation of this mRNA was thus identified: Thisprotein, designated MLL, is composed of 2709 aa. It is identified asnumber SEQ ID No. 75 in the attached sequence listing.

This sequence GS-N23 presents at least 86% homology with the followingsequences:

-   -   GS-N47: a 14,255-bp mRNA identified as number SEQ ID No. 48 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number L04731.

This mRNA sequence does not have a coding sequence. It comprises thesequence GS-N23 with a homology of 90%.

-   -   GS-N48: a 11,910-bp mRNA identified as number SEQ ID No. 49 in        the attached sequence listing. A BLAST search of the GENBANK        database identified it as accession number NM_(—)005933.

The mRNA sequence identified by the number SEQ ID No. 48 has a codingsequence from nucleotide 1 to nucleotide 11910. A protein GS-P43resulting from the translation of this mRNA was thus identified. Thisprotein, designated MLL, is composed of 3969 aa. It is identified by thenumber SEQ ID No. 99 in the attached sequence listing.

-   -   GS-N24: a 10,330-bp mRNA identified by the number SEQ ID No. 25        in the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number U72937        (DNA-dependent helicase and ATPase (ATRX), product 2 of the        alternative splice).

This sequence is homologous with at least 95% identity of the sequence:

-   -   GS-N49: a 10,452-bp mRNA identified by the number SEQ ID No. 50        in the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number U72936.

The mRNA sequence identified by the number SEQ ID No. 25 has a codingsequence from nucleotide 216 to nucleotide 7694. A protein GS-P24resulting from the translation of this mRNA was thus identified. Thisprotein, designated ATRX product 2, is composed of 2492 aa. It isidentified by the number SEQ ID No. 76 in the attached sequence listing.

The mRNA sequence identified by the number SEQ ID No. 50 has a codingsequence from nucleotide 950 to nucleotide 7816. A protein GS-P49resulting from the translation of this mRNA was thus identified. Thisprotein, designated ATRX product 1, is composed of 2288 aa. It isidentified by the number SEQ ID No. 100 in the attached sequencelisting.

The proteins identified by the numbers SEQ ID No. 76 and 100 arehomologous at the level of 90%.

-   -   GS-N25: a 1777-bp mRNA identified by the number SEQ ID No. 26 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number NM_(—)006476        (sialic acid transporter-CMP, member 1 (SLC3SA1).

The sequence of this mRNA has a coding sequence from nucleotide 28 tonucleotide 1041. A protein GS-P25 resulting from the translation of thismRNA was thus identified. This protein, designated sialic acidtransporter-CMP is composed of 337 aa. It is identified by the numberSEQ ID No. 77 in the attached sequence listing.

-   -   GS-N50: a 1874-bp mRNA identified by the number SEQ ID No. 225        in the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number BC008372.        The mRNA sequence identified by the number SEQ ID No. 225 does        not have a coding sequence.    -   GS-N26: a 3982-bp mRNA identified by the number SEQ ID No. 27 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number U26710.

The sequence of this mRNA has a coding sequence from nucleotide 323 tonucleotide 3271. A protein GS-P26 resulting from the translation of thismRNA was thus identified. This protein, designated Cbl-b, is composed of982 aa. It is identified by the number SEQ ID No. 78 in the attachedsequence listing.

-   -   GS-N27: a 3385-bp mRNA identified by the number SEQ ID No. 28 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number XM 39529.

This sequence presents 86% homology with the following sequence:

A 4461-bp cDNA (DKFZp564D173) identified by the number SEQ ID No. 51 inthe attached sequence listing. A BLAST search of the GENBANK sequencedatabase identified it as accession number AL110212. There is nocorresponding coding sequence.

The mRNA sequence identified by the number SEQ ID No. 27 has a codingsequence from nucleotide 107 to nucleotide 451. A protein GS-P27resulting from the translation of this mRNA was thus identified. Thisprotein, designated histone H2A.F/Z variant (H2AV), is composed of 114aa. It is identified by the number SEQ ID No. 79 in the attachedsequence listing.

-   -   GS-N28: a 1128-bp mRNA identified as number SEQ ID No. 29 in the        attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        NM_(—)001320.

This sequence has 77% homology with the sequence identified under theaccession number M30448 in the GENBANK database and identified by numberSEQ ID No. 289 in the attached sequence listing.

The sequence of this mRNA has a coding sequence from nucleotide 97 tonucleotide 744. A protein GS-P28 resulting from the translation of thismRNA was thus identified. This protein, designated casein kinase II,subunit beta, is composed of 215 aa. It is identified by the number SEQD No. 80 in the attached sequence listing.

-   -   GS-N29: a 18,207-bp mRNA identified by the number SEQ ID No. 30        in the attached sequence listing. A BLAST search in the GENBANK        sequence database identified it as accession number AF 156100.

The sequence of this mRNA has a coding sequence from nucleotide 230 tonucleotide 17140. A protein GS-P29 resulting from the translation ofthis mRNA was thus identified. This protein, designated hemicentine, iscomposed of 5636 aa. It is identified by the number SEQ ID No. 81 in theattached sequence listing.

This sequence GS-29 comprises the sequence GS-N52 below, presenting ahomology of 99% with it.

GS-N52: a 8546-bp mRNA identified by the number SEQ ID No. 52 in theattached sequence listing. A BLAST search of the GENBANK sequencedatabase identified it as accession number AJ306906.

The sequence of this mRNA has a sequence coding for a protein GS-P52 of2673 aa identified by the number SEQ ID No. 101 in the attached sequencelisting.

-   -   GS-N30: a 4325-bp mRNA identified by the number SEQ ID No. 31 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        NM_(—)015180.

The sequence of this mRNA has a coding sequence from nucleotide 123 tonucleotide 3041. A protein GS-P30 resulting from the translation of thismRNA was thus identified. This protein, designated SYNE-2, is composedof 1092 aa. It is identified by the number SEQ ID No. 82 in the attachedsequence listing.

-   -   GS-N31: a 4248-bp mRNA identified by the number SEQ ID No. 32 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number AF261758.

The sequence of this mRNA has a coding sequence from nucleotide 100 tonucleotide 1650. A protein GS-P31 resulting from the translation of thismRNA was thus identified. This protein, designated seladine-1, iscomposed of 516 aa. It is identified as number SEQ ID No. 83 in theattached sequence listing.

This mRNA sequence identified as number SEQ ID No. 32 in the attachedsequence listing is homologous with a 4187-bp sequence. A BLAST searchof the GENBANK sequence database identified it as accession numberD13643. It is identified under the number SEQ ID No. 53 in the attachedsequence listing.

This mRNA presents a partial coding sequence. A protein GS-P53 of 528 aaresulting from the translation of this mRNA was thus identified. It isidentified as number SEQ ID No. 102 in the attached sequence listing.

-   -   GS-N32: a 7764-bp mRNA identified by the number SEQ ID No. 33 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        NM_(—)001271.

The sequence of this mRNA has a coding sequence from nucleotide 708 tonucleotide 5927. A protein GS-P32 resulting from the translation of thismRNA was thus identified. This protein, designated CHD2, is composed of1739 aa. It is identified by the number SEQ ID No. 84 in the attachedsequence listing.

-   -   GS-N33: a 4693-bp mRNA identified by number SEQ ID No. 34 in the        attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number XM_(—)04255.

The sequence of this mRNA has a coding sequence from nucleotide 1702 tonucleotide 4107. A protein GS-P33 resulting from the translation of thismRNA was thus identified. This protein, designated BRD2, is composed of801 aa. It is identified by the number SEQ D No. 85 in the attachedsequence listing.

-   -   GS-N34: a 2983-bp mRNA identified by the number SEQ ID No. 35 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number BC007429.

The sequence of this mRNA has a coding sequence from nucleotide 200 tonucleotide 1069. A protein GS-P34 resulting from the translation of thismRNA was thus identified. This protein, designated syntaxin 3A, iscomposed of 289 aa. It is identified by the number SEQ ID No. 86 in theattached sequence listing.

-   -   GS-N35: a 12,227-bp mRNA identified by the number SEQ ID No. 36        in the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        NM_(—)015001.

The sequence of this mRNA has a coding sequence from nucleotide 205 tonucleotide 11,199. A protein GS-P35 resulting from the translation ofthis mRNA was thus identified. This protein, designated SHARP, iscomposed of 3664 aa. It is identified by the number SEQ ID No. 87 in theattached sequence listing.

-   -   GS-N36: a 5376-bp mRNA identified by the number SEQ ID No. 37 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number AF352051.

The sequence of this mRNA has a coding sequence from nucleotide 92 tonucleotide 4692. A protein GS-P36 resulting from the translation of thismRNA was thus identified. This protein, designated proliferationpotential-related protein, is composed of 1616 aa. It is identified asnumber SEQ ID No. 88 in the attached sequence listing.

This sequence identified by the number SEQ ID No. 37 presents 92%homology with the sequence coding for the protein RPBB6(retinoblastoma-binding protein 6) of 948 aa identified by the numberSEQ ID No. 288 in the attached sequence listing. The nucleotide sequencecorresponding to this protein comprises 0.2994 bp and the accessionnumber NM_(—)006910 in the GENBANK sequence database.

-   -   GS-N37: a 6626-bp mRNA identified by the number SEQ ID No. 38 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        XM_(—)005338.

The sequence of this mRNA has a coding sequence from nucleotide 245 tonucleotide 2989. A protein GS-P37 resulting from the translation of thismRNA was thus identified. This protein, designated protein HIP1, iscomposed of 914 aa. It is identified by the number SEQ D No. 89 in theattached sequence listing.

-   -   GS-N38: a 2366-bp mRNA identified by the number SEQ ID No. 39 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number BC000335.

The sequence of this mRNA has a coding sequence from nucleotide 12 tonucleotide 2237. A protein GS-P38 resulting from the translation of thismRNA was thus identified. This protein, designated nucleoporin 88 kDa,is composed of 741 aa. It is identified by the number SEQ ID No. 90 inthe attached sequence listing.

-   -   GS-N39: a 1543-bp mRNA identified by the number SEQ ID No. 40 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        XM_(—)049486.

The sequence of this mRNA has a coding sequence from nucleotide 86 tonucleotide 412. A protein GS-P39 resulting from the translation of thismRNA was thus identified. This protein, designated FK506 bindingprotein, is composed of 108 aa. It is identified by the number SEQ IDNo. 91 in the attached sequence listing.

-   -   GS-N40: a 3824-bp mRNA identified by the number SEQ ID No. 41 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        XM_(—)042827.

The sequence of this mRNA has a coding sequence from nucleotide 115 tonucleotide 3663. A protein GS-P40 resulting from the translation of thismRNA was thus identified. This protein, designated SALF protein, iscomposed of 1182 aa. It is identified by the number SEQ ID No. 92 in theattached sequence listing.

-   -   GS-N41: a 1365-bp mRNA identified by the number SEQ ID No. 42 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number BC010862.

The sequence of this mRNA has a coding sequence from nucleotide 277 tonucleotide 758. A protein GS-P41 resulting from the translation of thismRNA was thus identified. This protein is composed of 243 aa and isidentified by the number SEQ ID No. 93 in the attached sequence listing.

-   -   GS-N42: a 6147-bp mRNA identified by the number SEQ ID No. 43 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number        NM_(—)013390.

The sequence of this mRNA has a coding sequence from nucleotide 149 tonucleotide 4300. A protein GS-P42 resulting from the translation of thismRNA was thus identified. This protein, designated TMEM2, is composed of1383 aa. It is identified by the number SEQ ID No. 94 in the attachedsequence listing.

-   -   GS-N43: a 4357-bp mRNA identified by the number SEQ ID No. 44 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number AB029316.

The sequence of this mRNA has a coding sequence from nucleotide 318 tonucleotide 2834. A protein GS-P43 resulting from the translation of thismRNA was thus identified. This protein, designated Dorfin, is composedof 838 aa. It is identified by the number SEQ ID No. 95 in the attachedsequence listing.

-   -   GS-N44: a 1801-bp mRNA identified by the number SEQ ID No. 45 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it under the accession number        XM_(—)032382 (member 2 of the transmembranous superfamily 4).

The sequence of this mRNA has a coding sequence from nucleotide 81 tonucleotide 815. A protein GS-P44 resulting from the translation of thismRNA was thus identified. This protein, designated TM4SF2, is composedof 244 aa. It is identified by the number SEQ ID No. 96 in the attachedsequence listing.

-   -   GS-N45: a 2801-bp mRNA identified by the number SEQ ID No. 46 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number HSA133133.

The sequence of this mRNA has a coding sequence from nucleotide 184 tonucleotide 1737. A protein GS-P45 resulting from the translation of thismRNA was thus identified. This protein, designated Ecto-ATPdiphosphohydralase I, is composed of 517 aa. It is identified by numberSEQ ID No. 97 in the attached sequence listing.

CD39 and ecto-ATPDASE I could be two forms resulting from thealternative splicing of the same gene. The vector coding for theantisense oligonucleotide used in our study can inhibit the expressionof both CD39 and ecto-ATPDase I.

The results obtained with the vector GS-V45 in the framework show thatCD39 plays a direct role in angiogenesis.

It should also be noted that no role has been described to date forecto-ATPDase I in the regulation of angiogenesis.

-   -   GS-N46: a 4332-bp mRNA identified by the number SEQ ID No. 47 in        the attached sequence listing. A BLAST search of the GENBANK        sequence database identified it as accession number AJ306399.

The sequence of this mRNA has a coding sequence from nucleotide 56 tonucleotide 1828. A protein GS-P46 resulting from the translation of thismRNA was thus identified. This protein is composed of 590 aa. It isidentified by the number SEQ ID No. 98 in the attached sequence listing.

The expression of the mRNAs identified above is observed in humanendothelial cells that form capillary tubes. We thus demonstrate thatthe differential expression of the gene corresponding to each of thesemRNA accompanies the formation of neovessels by the endothelial cells.

Moreover, it is demonstrated herein that the induction of expression ofthe genes during angiogenesis is sensitive to the presence of differentinhibitors. In fact, when human endothelial cells forming neovessels arestimulated by an angiogenic factor (indicated in column 2 of table II),one observes an elevated expression of this mRNA whereas when the samehuman endothelial cells are stimulated by the same angiogenic factor andbrought into the presence of an anti-angiogenic factor (indicated incolumn 3 of table II) (where angiogenesis is inhibited) one observesthat the expression of this gene is also inhibited.

TABLE II Inducers of Inhibitors of SEQ. ID expression expression SEQ IDNo 1 (GS-N1) VEGF PF4 SEQ ID No 2 (GS-N2) VEGF PF4 SEQ ID No 3 (GS-N3)FGF2 IFN-gamma SEQ ID No 4 (GS-N4) VEGF IFN-gamma SEQ ID No 5 (GS-N5)VEGF IFN-gamma SEQ ID No 6 (GS-N6) VEGF IFN-gamma SEQ ID No 7 (GS-N7)VEGF TNF-alpha SEQ ID No 8 (GS-N8) VEGF TNF-alpha SEQ ID No 9 (GS-N9)VEGF IFN-gamma SEQ ID No 10 (GS-N10) VEGF IFN-gamma SEQ ID No 11(GS-N11) VEGF IFN-gamma SEQ ID No 12 (GS-N12) VEGF IFN-gamma SEQ ID No13 (GS-N13) VEGF IFN-gamma SEQ ID No 14 (GS-N14) VEGF IFN-gamma SEQ IDNo 15 (GS-N15) VEGF IFN-gamma SEQ ID No 17 (GS-N16) VEGF PF4 SEQ ID No18 (GS-N17) VEGF TSP-1 SEQ ID No 19 (GS-N18) VEGF PF4 SEQ ID No 20(GS-N19) VEGF PF4 SEQ ID No 21 (GS-N20) VEGF PF4 SEQ ID No 22 (GS-N21)VEGF PF4 SEQ ID No 23 (GS-N22) VEGF PF4 SEQ ID No 24 (GS-N23) FGF2TNF-alpha SEQ ID No 25 (GS-N24) VEGF IFN-gamma SEQ ID No 26 (GS-N25)VEGF IFN-gamma SEQ ID No 27 (GS-N26) VEGF IFN-gamma SEQ ID No 28(GS-N27) VEGF IFN-gamma SEQ ID No 29 (GS-N28) VEGF IFN-gamma SEQ ID No30 (GS-N29) FGF2 Ang-2 SEQ ID No 31 (GS-N30) VEGF TNF-alpha SEQ ID No 32(GS-N31) VEGF IFN-gamma SEQ ID No 33 (GS-N32) VEGF TNF-alpha SEQ ID No34 (GS-N33) VEGF IFN-gamma SEQ ID No 35 (GS-N34) VEGF TNF-alpha SEQ IDNo 36 GS-N35) VEGF IFN-gamma SEQ ID No 37 (GS-N36) VEGF IFN-gamma SEQ IDNo 38 (GS-N37) FGF2 Ang-2 SEQ ID No 39 (GS-N38) VEGF IFN-gamma SEQ ID N°40 (GS-N39) VEGF IFN-gamma SEQ ID No 41 (GS-N40) VEGF IFN-gamma SEQ IDNo 42 (GS-N41) VEGF IFN-gamma SEQ ID No 43 (GS-N42) VEGF Ang-2 SEQ ID No44 (GS-N43) FGF2 PF4 SEQ ID No 45 (GS-N44) FGF2 IFN-gamma SEQ ID No 46(GS-N45) VEGF IFN-gamma SEQ ID No 47 (GS-N46) FGF2 TNF-alpha SEQ ID No48 (GS-N47) FGF2 TNF-alpha SEQ ID No 49 (GS-N48) FGF2 TNF-alpha SEQ IDNo 50 (GS-N49) VEGF IFN-gamma SEQ ID No 51 (GS-N51) VEGF IFN-gamma SEQID No 52 (GS-N52) FGF2 Ang-2 SEQ ID No 53 (GS-N53) VEGF TNF-alpha SEQ IDNo 225 (GS-N50) VEGF IFN-gamma SEQ ID No 16 (GS-N54) VEGF IFN-gamma

It thus appears that there exists a direct correlation between theexpression of each of the genes GS-N1 to GS-N54 and the angiogenic stateof the human endothelial cells. 6.2 Verification of the role of theidentified genes in the regulation of angiogenesis.

Moreover, the functional role of the above-described genes in theformation of neovessels by human endothelial cells has been demonstratedherein.

In fact, an oligonucleotide specific of each of the identified genes,selected from among the oligonucleotides identified by the sequences SEQID No. 103 to SEQ ID No. 148 in the attached sequence listing, wasintroduced into the expression vector pCI-neo vector in the antisenseorientation.

The resultant vectors, designated GS-V1 to GS-V46 and identified bytheir sequence SEQ ID No. 149 to SEQ ID No. 194 in the attached sequencelisting, were used to repress the expression of the gene coding for thismRNA in human endothelial cells subsequent to the transfection of thesecells by this vector.

The human endothelial cells were then stimulated by the angiogenicfactors. The results obtained for each of the sequences illustratedbelow, using the antisense sequences and the corresponding vectors,indicated in Table III, shows that:

-   -   the repression of the expression of the genes SEQ ID No. 1 to        SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 to SEQ        ID No. 15, SEQ ID No. 17 to SEQ ID No. 53 and SEQ ID No. 225        inhibit the formation of neovessels by the human endothelial        cells; and that    -   the repression of the genes SEQ ID No. 6, SEQ ID No. 8, SEQ ID        No. 10 and SEQ ID No. 16 stimulates the formation of neovessels        by the human endothelial cells despite the presence of the        different angiogenic factors.

These results are also illustrated in the attached FIGS. 1 to 11.

TABLE III Genes Proteins Antisense Vector with Control Name SEQ. ID SEQ.ID sequences antisense inserted Fig. Fig. 1 SEQ ID No 1 SEQ ID No 54 SEQID No 103 SEQ ID No 149 1A 1F (GS-N1) (GS-P1) (257pb) (GS-V1)Angioinducine 2 SEQ ID No 2 SEQ ID No 55 SEQ ID No 104 SEQ ID No 150 1B1F (GS-N2) (GS-P2) (202pb) (GS-V2) Angiodockine 3 SEQ ID No 3 SEQ ID No56 SEQ ID No 105 SEQ ID No 151 2A 2C (GS-N3) (GS-P3) (242 bp) (GS-V3)Angioblastine 4 SEQ ID No 4 SEQ ID No 57 SEQ ID No 106 SEQ ID No 152 1C1F (GS-N4) (GS-P4) (211 bp) (GS-V4) Angioreceptine 5 SEQ ID No 5 SEQ IDNo 58 SEQ ID No 107 SEQ ID No 153 1D 1F (GS-N5) (GS-P5) (191 bp) (GS-V5)Angiodensine 6 SEQ ID No 6 SEQ ID No 59 SEQ ID No 108 SEQ ID No 154 3A3D (GS-N6) (GS-P6) (238 bp) (GS-V6) Vassoserpentine 7 SEQ ID No 7 SEQ IDNo 60 SEQ ID No 109 SEQ ID No 155 4A 4F (GS-N7) (GS-P7) (205 bp) (GS-V7)Angiosulfatine 8 SEQ ID No 8 SEQ ID No 61 SEQ ID No 110 SEQ ID No 156 3B3D (GS-N8) (GS-P8) (186 bp) (GS-V8) Vassoreceptine 9 SEQ ID No 9 SEQ IDNo 62 SEQ ID No 111 SEQ ID No 157 4B 4F (GS-N9) (GS-P9) (223 pb) (GS-V9)Angiokinasine 10 SEQ ID No 10 SEQ ID No 63 SEQ ID No 112 SEQ ID No 1583C 3D (GS-N10) (GS-P10) (247 pb) (GS-V10) Vassosubstratine 11 SEQ ID No11 SEQ ID No 64 SEQ ID No 113 SEQ ID No 159 4C 4F (GS-N11) (GS-P11) (162pb) (GS-V11) Angiosignaline 12 SEQ ID No 12 SEQ ID No 65 SEQ ID No 114SEQ ID No 160 4D 4F (GS-N12) (GS-P12) (166 bp) (GS-V12) Angiofoculine 13SEQ ID No 13 SEQ ID No 66 SEQ ID No 115 SEQ ID No 161 2B 2C (GS-N13)(GS-P13) (135 bp) (GS-V13) Angiohélicine 14 SEQ ID No 14 — SEQ ID No 116SEQ ID No 162 4E 4F (GS-N14) (136 bp) (GS-V14) 15 SEQ ID No 15 SEQ ID No67 SEQ ID No 117 SEQ ID No 163 1A 1F (GS-N15) (GS-P15) (152 bp) (GS-V15)Angioacyline 16 SEQ ID No 16 SEQ ID No 112 SEQ ID No 158 3C 3D (GS-N54)(247 bp) (GS-V10) 17 SEQ ID No 17 SEQ ID No 68 SEQ ID No 118 SEQ ID No164 5A 5F (GS-N16) (GS-P16) (417 bp) (GS-V16) PDCL 18 SEQ ID No 18 SEQID No 69 SEQ ID No 119 SEQ ID No 165 5B 5F (GS-N17) (GS-P17) (244 bp)(GS-V17) RPL3 19 SEQ ID No 19 SEQ ID No 70 SEQ ID No 120 SEQ ID No 1665C 5F (GS-N18) (GS-P18) (311 bp) (GS-V18) homol.RNF20 20 SEQ ID No 20SEQ ID No 71 SEQ ID No 121 SEQ ID No 167 5D 5F (GS-N19) (GS-P19) (246bp) (GS-V19) homol.SFRS4 21 SEQ ID No 21 SEQ ID No 72 SEQ ID No 122 SEQID No 168 10A 10F (GS-N20) (GS-P20) (203 bp) (GS-V20) CPD 22 SEQ ID No22 SEQ ID No 73 SEQ ID No 123 SEQ ID No 169 5E 5F (GS-N21) (GS-P21) (253bp) (GS-V21) USP9X 23 SEQ ID No 23 SEQ ID No 74 SEQ ID No 124 SEQ ID No170 6A 6F (GS-N22) (GS-P22) (173 bp) (GS-V22) NRD1 24 SEQ ID No 24 SEQID No 75 SEQ ID No 125 SEQ ID No 171 10B 10F (GS-N23) (GS-P23) (228 bp)(GS-V23) Homol. HRX, ALL-1, MLL 25 SEQ ID No 25 SEQ ID No 76 SEQ ID No126 SEQ ID No 172 6B 6F (GS-N24) (GS-P24) (381 bp) (GS-V24) ATRX 26 SEQID No 26 SEQ ID No 77 SEQ ID No 127 SEQ ID No 173 6C 6F (GS-N25)(GS-P25) (395 bp) (GS-V25) transp.ac sial.-CMP1 27 SEQ ID No 27 SEQ IDNo 78 SEQ ID No 128 SEQ ID No 174 6D 6F (GS-N26) (GS-P26) (381 bp)(GS-V26) CBL-b 28 SEQ ID No 28 SEQ ID No 79 SEQ ID No 129 SEQ ID No 1756E 6F (GS-N27) (GS-P27) (298 bp) (GS-V27) 29 SEQ ID No 29 SEQ ID No 80SEQ ID No 130 SEQ ID No 176 7A 7F (GS-N28) (GS-P28) (413 bp) (GS-V28)CSNK2B 30 SEQ ID No 30 SEQ ID No 81 SEQ ID No 131 SEQ ID No 177 7B 7F(GS-N29) (GS-P29) (564 bp) (GS-V29) Hémicentine 31 SEQ ID No 31 SEQ IDNo 82 SEQ ID No 132 SEQ ID No 178 7C 7F (GS-N30) (GS-P30) (414 bp)(GS-V30) SYNE-2 32 SEQ ID No 32 SEQ ID No 83 SEQ ID No 133 SEQ ID No 1797D 7F (GS-N31) (GS-P31) (298 bp) (GS-V31) Séladine-1 33 SEQ ID No 33 SEQID No 84 SEQ ID No 134 SEQ ID No 180 7E 7F (GS-N32) (GS-P32) (365 bp)(GS-V32) CHD2 34 SEQ ID No 34 SEQ ID No 85 SEQ ID No 135 SEQ ID No 1818A 8F (GS-N33) (GS-P33) (270 bp) (GS-V33) BRD2 35 SEQ ID No 35 SEQ ID No86 SEQ ID No 136 SEQ ID No 182 8B 8F (GS-N34) (GS-P34) (298 bp) (GS-V34)Syntaxine 3A 36 SEQ ID No 36 SEQ ID No 87 SEQ ID No 137 SEQ ID No 183 8C8F GS-N35) (GS-P35) (117 bp) (GS-V35) SHARP 37 SEQ ID No 37 SEQ ID No 88SEQ ID No 138 SEQ ID No 184 10C 10F (GS-N36) (GS-P36) (96 bp) (GS-V36)PLPP 38 SEQ ID No 38 SEQ ID No 89 SEQ ID No 139 SEQ ID No 185 8D 8F(GS-N37) (GS-P37) (393 bp) (GS-V37) HIP1 39 SEQ ID No 39 SEQ ID No 90SEQ ID No 140 SEQ ID No 186 8E 8F (GS-N38) (GS-P38) (100 bp) (GS-V38)NUP88 40 SEQ ID N° 40 SEQ ID N° 91 SEQ ID No 141 SEQ ID No 187 10D 10F(GS-N39) (GS-P39) (90 bp) (GS-V39) FKPB1A 41 SEQ ID No 41 SEQ ID N° 92SEQ ID No 142 SEQ ID No 188 9A 9F (GS-N40) (GS-P40) (144 bp) (GS-V40)SALF 42 SEQ ID No 42 SEQ ID N° 93 SEQ ID No 143 SEQ ID No 189 10E 10F(GS-N41) (GS-P41) (113 bp) (GS-V41) Homol.P29 43 SEQ ID No 43 SEQ ID N°94 SEQ ID No 144 SEQ ID No 190 9B 9F (GS-N42) (GS-P42) (180 bp) (GS-V42)TMEM2 44 SEQ ID No 44 SEQ ID N° 95 SEQ ID No 145 SEQ ID No 191 9C 9F(GS-N43) (GS-P43) (507 bp) (GS-V43) Dorfine 45 SEQ ID No 45 SEQ ID N° 96SEQ ID No 146 SEQ ID No 192 9D 9F (GS-N44) (GS-P44) (632 bp) (GS-V44)TM4SF2 46 SEQ ID No 46 SEQ ID N° 97 SEQ ID No 147 SEQ ID No 193 9E 9F(GS-N45) (GS-P45) (704 bp) (GS-V45) Ecto-ATPase I 47 SEQ ID No 47 SEQ IDN° 98 SEQ ID No 148 SEQ ID No 194 11A 11B (GS-N46) (GS-P46) (257 bp)(GS-V46) Sélénoprotéine N 48 SEQ ID No 48 — SEQ ID No 125 SEQ ID No 17110B 10F (GS-N47) (228 bp) (GS-V23) 49 SEQ ID No 49 SEQ ID N° 99 SEQ IDNo 125 SEQ ID No 171 10B 10F (GS-N48) (GS-P48) (228 bp) (GS-V23) MLL 50SEQ ID No 50 SEQ ID N° 100 SEQ ID No 126 SEQ ID No 172 6B 6F (GS-N49)(GS-P49) (381 bp) (GS-V24) ATRX 51 SEQ ID No 51 — SEQ ID No 129 SEQ IDNo 175 6E 6F (GS-N51) (298 bp) (GS-V27) 52 SEQ ID No 52 SEQ ID N° 101SEQ ID No 131 SEQ ID No 177 7B 7F (GS-N52) (GS-P52) (564 bp) (GS-V29)Fibuline 6 53 SEQ ID No 53 SEQ ID N° 102 SEQ ID No 133 SEQ ID No 179 7D7F (GS-N53) (GS-P53) (298 bp) (GS-V31) Séladine 1 54 SEQ ID No 225 — SEQID No 127 SEQ ID No 173 6C 6F (GS-N50) (395 bp) (GS-V25)

The invention claimed is:
 1. A composition comprising: a carrierselected from the group consisting of saline solutions, physiologicalsaline, phosphate buffered saline (PBS); polyethylene glycols,glycerine, propylene glycol, synthetic solvents; antibacterial agents,benzyl alcohol, methyl parabens; antioxidants, ascorbic acid, sodiumbisulfite; chelating agents, ethylenediaminetetraacetic acid; buffers,acetates, citrates, or phosphates, agents for the adjustment oftonicity, sodium chloride, dextrose; stabilizing or preservative agents,sodium bisulfite, sodium sulfite, ascorbic acid, citric acid and itssalts, ethylenediaminetetraacetic acid, benzalkonium chloride, methyl-or propylparaben chlorobutanol; and combinations thereof; and anantisense sequence consisting of SEQ ID NO:
 107. 2. An antisensenucleotide sequence consisting of SEQ ID NO:
 107. 3. A mammalianexpression vector comprising the antisense sequence of claim
 2. 4. Avector GS-V5 identified as SEQ ID NO: 153 comprising the antisensesequence of the nucleotide sequence SEQ ID NO: 5 comprising SEQ ID NO:107.